Barkhorn flew his first combat sorties over Belgium and France during the Battle of France, and later over England during the Battle of Britain, flying the Messerschmitt Bf 109E. He did not have any success at shooting down the enemy, although he flew many combat sorties and was shot down into the English Channel twice. On 1 August 1940, Barkhorn was transferred to 6./JG 52. Shortly thereafter he was rewarded with the Eisernes Kreuz erster Klasse. In Staffel, he flew alongside another promising pilot, Hans-Joachim Marseille.

In 1941, JG 52 was transferred to the east and participated in Operation Barbarossa, the invasion of the Soviet Union on 21 June 1941. Not long after, Gerhard Barkhorn made his first victory, shooting down a Red Air Force aircraft on 2 July 1941, while flying his 120th combat sortie. This victory seemed to have a reassuring effect on Barkhorn, who finally had found his 'shooting eye'. By November, his tally had reached 10 victories, and he was promoted to Oberleutnant on 11 November 1941.

On 21 May 1942, Barkhorn was appointed Staffelkapitän of 4./JG 52. He continued to score victories over the next year, until on 19 July 1942, he became "ace in one day" by shooting down 6 aircraft in his Bf 109F. However he was wounded on 25 July and was put out of action for two months, returning to combat in October. On 23 August 1942, Barkhorn received the Ritterkreuz for having shot down a total of 64 aircraft. On 19 December 1942, Barkhorn reached 100 victories, a feat for which he was awarded Eichenlaub to his Ritterkreuz on 11 January 1943. During this period, JG 52 switched to the Messerschmitt Bf 109G-6 aircraft.

Gerhard Barkhorn, now a Hauptmann, was appointed Gruppenkommandeur of II./JG 52 on 1 September 1943. By this time he had over 150 victories under his belt, and he reached his 200th victory on 30 November 1943. By 13 February 1944, he reached 250 victories, and a price was put on his head by the Red Air Force. Shortly thereafter, on 2 March, he was given the Schwerter to his Ritterkreuz. He attended the wedding of fellow ace Erich Hartmann as the best man. Barkhorn was promoted to Major on 1 May 1944.

On 31 May 1944, Barkhorn was shot down by a Russian P-39 Airacobra. He managed to crash-land behind his own lines but was severely wounded and was hospitalized for four months. Returning to the fray in October, he claimed his 275th victory on 14 November. Barkhorn scored his 301st and final victory on 5 January 1945.

On 16 January 1945, Barkhorn was assigned as Geschwaderkommodore of Jagdgeschwader 6, a unit assigned to defence of the Reich equipped with the Focke-Wulf Fw-190D (although Barkhorn preferred to keep his Bf 109G-6 fighter as well). JG 6 was a unit mostly consisting of new recruits and former Bf-110 pilots and suffered heavy losses against the American air fleets. Barkhorn did not last long in this position and was forced to take a medical absence because of severe physical and mental strain.

After his hospitalization, Barkhorn was invited by Adolf Galland to join the elite Jagdverband 44 flying the Messerschmitt Me-262 jet-fighter. He found flying the Me 262 over the western front difficult, and he did not score any victories in it. On 21 April 1945, he flew his 1104th and last mission. The engine of his aircraft flamed out as he was approaching an enemy bomber formation, and he was forced to make an emergency landing. As he approached the airfield his plane was attacked by several prowling P-51 fighters, but Barkhorn managed to land his burning plane. He received a slight wound as a result of this action. Taken prisoner, he was released from Allied captivity in September 1945. Barkhorn claimed 301 victories in 1,104 operational sorties. His total included 110 Yak fighters, 87 LaGG fighters, 21 Il-2's and 12 twin-engined medium bombers. He was shot down 9 times, bailed out once and was wounded 3 times.

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