This study was carried out to investigate aflatoxin M₁ (AFM₁) in raw milk of ruminant animals (cows, buffaloes, sheep, goats and camels) in Luxor Governorate to know any of these species milk is contaminated by the toxin and to determine its concentration to avoid its harmful effect on consumers' health. A total number of 165 milk samples were collected from various villages at the main three cities in Luxor Governorate (Esna, Armant and Luxor cities) in winter season 2015-2016 (11 milk samples from each species per city) and the samples had been analyzed by ELIZA test kits. The obtained results revealed that AFM₁ levels were lower than previous surveys in Egypt. The percent of positive milk samples in all species were 32.7, 58.18 and 56.36% from Esna, Armant and Luxor cities, respectively. AFM₁ could not be detected in sheep and goat samples from Esna, camel and goat samples from Armant and camel samples from Luxor city. Overall, the percent of positive milk samples in all cities from Luxor Governorate were 66.6% (22 out 33) in cows, 63.6% (21 out 33) in buffaloes, 15.2% (5 out 33) in camels, 66.7% (22 out 33) in sheep and 33.3% (11 out 33) in goats. AFM₁ mean values in milk samples of cows, buffaloes, camels, sheep and goats were 4.518, 1.951, 0.091, 2.966 and 0.582 ng/l respectively. The highest mean value of AFM₁ (10.953 ng/l) was found in cow's milk from Armant followed by sheep milk from Luxor (6.811ng/l) then buffaloes milk from Armant (4.005 ng/l). The highest value of AFM₁ (14.307 ng/l) was detected in cow's milk from Armant city followed by (13.177 ng/l) in buffaloes milk from Luxor. Concerning the health hazard for consumers, no milk samples exceeded the permissible limits of the US regulations (500ng/l) and the European Commission regulations (50ng/l), while all positive samples of raw milk are exceeding Egyptian regulations (free from AFM₁). In conclusion, high prevalence of AFM₁ in milk from Luxor Governorate indicated that the contamination of raw milk is very high and this due to the contamination of feedstuffs of these animals with AFB₁. Because of these findings, we need to survey aflatoxins incidence and levels in feedstuffs and milk during all seasons of the year in this areas.