Milk fat content and fatty acid (FA) composition have great economic value to the dairy industry as they are directly associated with taste and chemical-physical characteristics of milk and dairy products. In addition, consumers' choices are not only based on the nutritional aspects of food, but also on products known to promote better health. Milk FA composition is also related to the metabolic status and physiological stages of cows and thus can also be used as indicator for other novel traits of interest (e.g., metabolic diseases and methane yield). Genetic selection is a promising alternative to manipulate milk FA composition. In this study, we aimed to (1) estimate time-dependent genetic parameters for 5 milk FA groups (i.e., short-chain, medium-chain, long-chain, saturated, and unsaturated) predicted based on milk mid-infrared spectroscopy, for Canadian Ayrshire and Jersey breeds, and (2) conduct a time-dependent, single-step genome-wide association study to identify genomic regions, candidate genes, and metabolic pathways associated with milk FA. We analyzed 31,709 test-day records of 9,648 Ayrshire cows from 268 herds, and 34,341 records of 11,479 Jersey cows from 883 herds. The genomic database contained a total of 2,330 Ayrshire and 1,019 Jersey animals. The average daily heritability ranged from 0.18 (long-chain FA) to 0.34 (medium-chain FA) in Ayrshire, and from 0.25 (long-chain and unsaturated FA) to 0.52 (medium-chain and saturated FA) in Jersey. Important genomic regions were identified in Bos taurus autosomes BTA3, BTA5, BTA12, BTA13, BTA14, BTA16, BTA18, BTA20, and BTA21. The proportion of the variance explained by 20 adjacent SNP ranged from 0.71% (saturated FA) to 1.11% (long-chain FA) in Ayrshire, and from 0.70% (unsaturated FA) to 3.09% (medium-chain FA) in Jersey cattle. Important candidate genes and pathways were also identified, such as the PTK2 and TRAPPC9 genes, associated with milk fat percentage, and HMGCS, FGF10, and C6 genes, associated with fertility traits and immune response. Our findings on the genetic parameters and candidate genes contribute to a better understanding of the genetic architecture of milk FA composition in Ayrshire and Jersey dairy cattle.