There is a growing interest in odd- and branched-chain fatty acids (OBCFA) in milk following reports that several branched-chain fatty acids (FA) have health promoting effects, and certain milk OBCFA could serve as a biomarker to assess ruminal function. Twenty-four Holstein cows were fed 3 low-forage diets containing 30 g/kg of dry matter of prilled palm fat (PPF), sunflower oil (SO), or an equal mixture of both fats (experiment 1) or 3 diets containing 30 g/kg of dry matter of SO with a forage-to-concentrate ratio of 39:61, 44:56, or 48:52 (Experiment 2); diets were fed to investigate milk OBCFA composition and to explore the relationships between ruminal VFA and milk OBCFA using principal component analysis. Including SO in diets decreased yields of milk 13:0 anteiso, 15:0 anteiso, 15:0, 17:0, cis-9 15:1, and cis-9 17:1 compared with PPF. The molar proportion of ruminal propionate was the lowest and the yields of milk 14:0 iso and 16:0 iso were the greatest with the diet containing both fat supplements. Replacing concentrate with forages linearly increased ruminal acetate and yields of milk 13:0 iso, 14:0 iso, 15:0 iso, 16:0 iso, 17:0 iso, 13:0 anteiso, 15:0 anteiso, 15:0, 17:0, cis-9 15:1, and cis-9 17:1. The principal component analysis revealed that ruminal molar proportion of acetate related to concentrations of milk iso FA containing <17-carbon, whereas ruminal propionate related to milk 15:0, 17:0, cis-9 15:1, and cis-9 17:1, with the stronger correlations between milk OBCFA and ruminal acetate than propionate. No associations were found between ruminal molar proportion of butyrate and milk OBCFA concentrations. The results suggest that complete replacement of PPF with SO at 30 g/kg of dry matter in low-forage diets is not an effective strategy to enhance bioactive branched-chain FA in milk, rather this feeding practice lowers anteiso FA in milk; however, increasing forage proportion in diets containing SO enhances several iso and anteiso FA in milk. The milk OBCFA concentrations have stronger correlations with ruminal acetate molar proportion than with propionate or butyrate in cows fed diets containing supplemental fats.