A study of irrigated pasture-based dairy farms that used split calving (autumn and spring) was undertaken in northern Victoria, Australia, to examine associations between nutrition, time of year and season of calving on the concentrations of isomers of trans 18  :  1 fatty acids and conjugated linoleic acids (CLA) in milk fat. Factors associated with time of year explained most of the variation, with the highest concentrations observed in spring and summer when pasture intake by herds was high. However, there was substantial variation observed between herds and time of year. The mean total CLA concentration was 9.1 mg/g milk fatty acids (range 1.1–35.4 mg/g) with the cis,trans-9,11 accounting for ~84% of the total CLA. The mean total trans 18 : 1 concentration was 60.5 mg/g milk fatty acids (range 13.6–267 mg/g) with vaccenic acid (trans-11 18 : 1) accounting for ~53% of total trans 18 : 1 fatty acids. Total CLA and vaccenic acid were highest in August–September (southern hemisphere spring) (15.1 and 76.3 mg/g milk fat) and lowest in November–March (5.6 mg/g milk fat) and May–July (9.53 mg/g milk fat), respectively. There was no association between season of calving and milk CLA or trans 18 : 1 fatty acid concentrations. Trans-10 and -11 18 : 1 fatty acids and trans/trans-CLA were negatively correlated with milk fat concentrations. Management strategies designed to increase the concentration of CLA and trans 18 : 1 fatty acids in milk fat would not need to consider the effects of season of calving or stage of lactation, but should focus on pasture availability and quality.