This trial aimed to study the changes in the yield, composition, and fatty acid (FA) profile of ewes’ milk during suckling and milking periods. Nineteen multiparous Najdi ewes were kept under identical management and feeding conditions during a 12-week trial that was divided into two periods. Milk samples were collected on each sampling day from the total yield during the suckling (3rd, 6th, and 9th week) and milking (12th week) periods. The milk yield and the total solids, fat, protein, and lactose contents of the milk remained constant throughout the suckling weeks, but the protein content increased and milk yield and lactose content decreased after weaning. There were no differences in milk saturated FA (SFA), unsaturated FA (UFA), and monounsaturated FA (MUFA) contents during the suckling and milking periods, whereas the polyunsaturated FA content increased during the milking period. There were no differences in the individual milk SFA during the weeks of suckling, but the contents of caproic acid (C6:0), caprylic acid (C8:0), margaric acid (C17:0), and stearic acid (C18:0) decreased. However, myristic acid (C14:0), lauric acid (C12:0), and isomers of pentadecanoic (C15:0) acid increased during the milking period. The percentage of vaccenic acid (C18:111t), rumenic acid (C18:29c,11t; conjugated linoleic acid (CLA)), ω-3 FA group, and the atherogenicity index did not differ during the suckling period, but increased after weaning in the 12th week of lactation. This study demonstrated that website weaning lambs in Najdi ewes is probably a factor that strongly affects milk traits by changing yield, composition, and FA profile.