Western Canadian bison meat is renowned for its natural healthfulness; however, studies on the dietary effects on the fatty acid (FA) profile are limited. Herein, we evaluated the FA profiles of retail bison (longissimus dorsi) representing grain-fed (Grain), grass-fed (Grass) and grass-fed plus oat and pea screening supplement from early (Early-con) or late (Late-con) season harvested finishing regimes. Bison meat contained less than 30 mg fat/g meat, and was lowest for Early-con bison. Proportions of polyunsaturated FA (PUFA) were greatest in Early-con and lowest for Late-con bison. Early-con bison had the greatest proportion of omega-6 (n-6) FA and Late-con bison the lowest, yet as mg/g meat, total n-6 content did not differ. In contrast, Grass and Early-con bison had greater proportions of 18:3n-3, 20:5n-3, 22:5n-3, 22:6n-3 and total omega-3 (n-3) FA. The n-3 content for Grain, Grass, Early-con and Late-con bison were 38, 90, 69 and 69 mg/100 g meat, respectively. The 3:1 n-6/n-3 ratios of Grass, Early-con and Late-con bison were superior to the 7:1 ratio of Grain bison. Proportions of potentially beneficial biohydrogenation intermediates (BI), including t11-18:1 and c9, t11-conjugated linoleic acid, were greater for Early-con and Late-con bison. Proportions of cis-monounsaturated FA were similar for both Grain and Grass bison; however, Late-con was greater than Early-con bison. Cumulatively, Grass, Early-con and Late-con bison were more desirable compared to Grain on account of greater proportions of n-3 FA and a lower n-6/n-3 ratio. Furthermore, seasonal supplementation enhanced the BI proportions with potential beneficial bioactivity in Early-con and Late-con bison.