No. 49 Squadron received the first Hampdens in September 1938 and by the end of the year, 49 and 83 Squadrons at RAF Scampton had re-equipped. A total of 226 Hampdens were in service with ten squadrons by the start of the Second World War, with six forming the operational strength of 5 Group of Bomber Command based in Lincolnshire. Despite its speed and manoeuvrability, the Hampden was no match for Luftwaffe fighters and its career as a day bomber was brief. Hampdens continued to operate at night on bombing raids over Germany and mine-laying (code-named "gardening") in the North Sea and the French Atlantic ports.
Flight Lieutenant Rod Learoyd of 49 Squadron, was awarded the Victoria Cross for the attack that he led on the Dortmund-Ems canal on 12 August 1940. Sergeant John Hannah was the wireless operator/air gunner of an 83 Squadron Hampden and was awarded the Victoria Cross on 15 September 1940, when he fought the flames of the burning aircraft, allowing the pilot to return it to base.
Almost half of the Hampdens built, 714, were lost on operations, taking with them 1,077 crew killed and another 739 missing. German flak accounted for 108, one hit a German barrage balloon, 263 Hampdens crashed because of "a variety of causes" and 214 others were classed as "missing". Luftwaffe pilots claimed 128 Hampdens, shooting down 92 at night. Guy Gibson spent most of the first two years of his wartime service flying Hampdens and his book Enemy Coast Ahead (1946) gives a strong flavour of the trials and tribulations of taking these aircraft into action. The last Bomber Command sorties by Hampdens were flown on the night of 14/15 September 1942 by 408 Squadron, RCAF. After being withdrawn from Bomber Command in 1942, it operated with RAF Coastal Command through 1943 as a long-range torpedo bomber, (the Hampden TB Mk I with a Mk XII torpedo in an open bomb bay and a 500-pound (230 kg) bomb under each wing) and as a maritime reconnaissance aircraft.
The Hampden was also used by the Royal Canadian Air Force (RCAF), Royal Australian Air Force (RAAF), Royal New Zealand Air Force (RNZAF), Aviatsiya Voenno-Morskogo Flota (AV-MF: Maritime Military Fleet Aviation) of the Soviet Union and the Swedish Flygvapnet (Air Force).
The Hampden in RCAF service included the 160 examples manufactured in Canada by the Victory Aircraft consortium. Of the total built, 84 were shipped by sea to Great Britain, while the remainder came to Patricia Bay (Victoria Airport) B.C., to set up No. 32 OTU (RAF) used for bombing and gunnery training. Typical exercises at 32 OTU consisted of patrolling up the West Coast of Vancouver Island at night or flying out into the Pacific to a navigational map coordinate, often in adverse and un-forecast inclement weather. Due to attrition from accidents, about 200 "war weary" Hampdens were later flown from the U.K. to Pat Bay as replacements.
In September 1942, the crews of 32 Hampdens from No. 144 Squadron RAF and No. 455 Squadron RAAF flew from Sumburgh in the Shetland Islands to Vaenga (renamed Severomorsk in 1951) in Murmansk Oblast, Russia, a hazardous route often subject to poor weather and spanning more than 2,100 nautical miles (3,900 km), partly over enemy-occupied territory in Norway and Finland. Nine Hampdens were lost en route. From Vaenga, 144 and 455 Squadrons escorted Arctic Convoy PQ 18. After the convoy arrived, the Wing's personnel returned by sea to the UK and the 23 surviving Hampdens were transferred to the Aviatsiya Voenno-Morskogo Flota. These Hampdens were then flown by the 3rd Squadron of 24 MTAP (??????-????????? ????????/Mine-torpedo aviation regiment [Anti-Shipping Wing]) until at least 1943. The Flygvapnet assigned an HP.52 to Reconnaissance Wing F 11 at Nyköping for evaluation, under the designation P5. After the war, the aircraft was sold to SAAB where it was used as an avionics testbed.