Frequencies of alleles at the DRB3.2 locus of the bovine major histocompatibility complex for two genetic lines of Holsteins were compared, and allelic effects on yield and udder health traits were examined. The DRB3.2 genotypes of 186 cows and heifers were determined using DNA that had been extracted from blood samples. The cattle were from a designed selection experiment for milk yield. After edits, 173 cows and heifers with known alleles remained (54 from a control line and 119 from a selection line for milk yield). A total of 19 alternative DRB3.2 alleles appeared across the two lines, and frequencies ranged from 0.3 to 21.4%. Allelic frequencies differed significantly between the selection and control lines. Alleles 3, 8, 9, 10, 26, and 28 were more frequent in the control line, but alleles 16, 24, and 27 were more frequent in the selection line. An animal model was used to estimate gene substitution effects of alternative alleles on milk yield, fat yield, and protein yield, SCS, chronically elevated SCS, acutely elevated SCS, and mastitis incidence. First, second, and third lactations were analyzed separately. Allele 7 was associated with a significant increase in protein yield during first and second lactations and a significant increase in chronically elevated SCS and acutely elevated SCS during second lactation. Because of conflicting results with other studies and a limited numbers of cows, additional research is needed before specific alleles are confirmed to be associated with measures of yield and udder health.