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Simple Summary: Cows are one of the oldest livestock that are bred for both meat and milk. There are only two local cow breeds in Latvia—Latvian Brown and Latvian Blue—which, until now, have not been genetically characterized. This study examined the genetic background of both local breeds in Latvia and identified genetic factors associated with mastitis, a common inflammation of the mammary gland that affects animal welfare and the quality of milk. Whole genome sequencing was performed on blood and semen samples, and the results from the obtained data show that both breeds had similar levels of genetic diversity compared with other breeds. The Latvian Blue breed showed a higher genetic variance compared with the Latvian Brown breed. Specific mastitis-associated genome regions were identified for each breed. This study provides fundamental insights into the genetics of local Latvian cow breeds and provides the first insight into the hereditary factors of mastitis in dairy cows. It may also facilitate the development of genetic tests to aid breeders in their attempts to maintain and improve local and foreign Latvian cattle breeds. Latvia has two local Bos taurus breeds—Latvian Brown (LBG) and Latvian Blue (LZG)—characterized by a good adaptation to the local climate, longevity, and high fat and protein contents in milk. Since these are desired traits in the dairy industry, this study investigated the genetic background of the LBG and LZG breeds and identified the genetic factors associated with mastitis. Blood and semen samples were acquired, and whole genome sequencing was then performed to acquire a genomic sequence with at least 35× or 10× coverage. The heterozygosity, nucleotide diversity, and LD analysis indicated that LBG and LZG cows have similar levels of genetic diversity compared to those of other breeds. An analysis of the population structure revealed that each breed clustered together, but the overall differentiation between the breeds was small. The highest genetic variance was observed in the LZG breed compared with the LBG breed. Our results show that SNP rs721295390 is associated with mastitis in the LBG breed, and SNPs rs383806754, chr29:43998719CG>C, and rs462030680 are associated with mastitis in the LZG breed. This study shows that local Latvian LBG and LZG breeds have a pronounced genetic differentiation, with each one suggesting its own mastitis-associated SNP profile. [ABSTRACT FROM AUTHOR]