This study examined the effects of milking frequency, breed, and stage of lactation on the milk fat globules (MFG) size and fatty acids (FA) composition of sheep milk. Milk from Karagouniko (n= 13) and Chios (n= 13) ewes was sampled postpartum on the 93rd, 101st, 108th, 121st, 156th, and 188th days of lactation. On the 108th day, the ewes were divided randomly into two milking groups: Once daily at 06:00 a.m. or twice daily at 06:00 a.m. and 16:00 p.m. Morphometric characteristics of MFG and FA composition were determined for each sample. Once versus twice daily milking had no effect on MFG dimensions, which tended to vary according to breed (smaller MFG were secreted from Chios withp= 0.065), while the stage of lactation had a significant effect (p< 0.001). FA composition differed significantly according to the stage of lactation and breed. The FA profile of the Karagouniko breed showed higher concentrations of short-chain FA. The milk samples from late lactation were characterized by higher concentrations of mono-unsaturated FA (MUFA) compared to early and mid-lactation. Moreover, correlations were found between the average diameter of MFG and FA concentrations, where the size of MFG was positively correlated with saturated FA (SFA) and negatively correlated with MUFA.