This paper investigated sources of variation of milk fatty acid (FA, g/100 g total FA) profile routinely predicted by mid-infrared spectroscopy in Pinzgauer dual-purpose cattle breed. A total of 19,578 individual milk samples collected from 1232 cows and 77 herds between 2011 and 2014 were available for phenotypic characterisation. Data were analysed using a linear mixed model which included stage of lactation, parity and their interaction as fixed effects, and cow and herdtest-day as random effects. Milk yield averaged 20.87 kg/d, and means for fat, protein and casein were 4.01, 3.44 and 2.72%, respectively. Saturated FA (SFA), monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) averaged 69.20, 25.43 and 3.19 g/100 g total FA, respectively. About half of the SFA were provided by C16:0 (31.24 g/100 g total FA), and almost all MUFA by C18:1 (22.18 g/100 g total FA). Fixed effects were significant in explaining the variation of the studied traits. Milk of first-parity cows had the lowest percentage of SFA, medium-chain FA and C14:0, and the greatest percentage of MUFA, PUFA, trans FA, short-and long-chain FA, and C18:1. Saturated FA, C14:0 and C16:0 increased from calving until 120 days in milk, whereas unsaturated FA, MUFA, PUFA, long-chain FA, C18:0 and C18:1 decreased. Although both parity and stage of lactation affected milk FA, greater variation was observed during lactation than among parities.