This study aimed to determine and compare the fatty acid composition and atherogenecity index of milk fat from Nguni, Boer and non-descript at two different sampling periods. The goats (n = 10 for each goat genotypes) were raised under an extensive system of husbandry. Milk samples were collected from each individual goat to determine fatty acids, as methyl esters, using gas chromatography. The result revealed significant differences (P < 0.05) in fatty acid profiles among the three goat genotypes for butyric, caproic, myristic, palmitic, stearic, vaccenic, linoleic, conjugated linoleic, α-linolenic, arachidic, eicosatrienoic and docosapentaenoic acids, among other milk fats. The time of milk sampling also had a significant effect (P < 0.05) on caproic, lauric, myristic, palmitic, palmitoleic, margaric, stearic, linoleic, arachidonic, docosapentaenoic and conjugated linoleic acids of milk fat. In addition, Nguni goats' milk fat had a healthier fatty acid than those of Boer and non-descript goats, characterized by a lower content in saturated fatty acid (68.65, 73.77 and 71.73 (P < 0.05), respectively). Goat genotypes and milk sampling time had an influence on the fatty acid composition of goat milk. These findings support the fact that goat milk is an important source of healthpromoting substances and support the sustainable use of animal genetic resources to improve goat milk fat quality, and hence deserve further investigation with local indigenous goat breeds.