An investigation was conducted to identify polymorphism in mannose-binding lectin 1 (MBL1) gene and its effect on udder health and performance traits in dairy cattle and buffalo of India. Candidate single-nucleotide polymorphism (SNP) c.2534G > A of MBL1 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). All the possible genotypes for SNP c.2534G > A (GG, AG, and AA) were observed in the studied population. However, Sahiwal cows revealed dimorphic pattern (AG and GG). The effect of targetedSNP on incidence of mastitis wasevaluated and found to be significant. Animals with GG genotype were less susceptible to clinical mastitis and had comparatively lower somatic cell score (SCS) in Hardhenu cattle (P < 0.01) and Murrah buffalo (P < 0.05). Animals having GG genotype also exhibited significantly (P < 0.05) lower age at first calving (AFC). AG genotyped Murrah buffalo animals revealed significantly higher second lactation milk yield (P < 0.01). GG genotype with SCS and AFC could therefore be exploited as a promising candidate marker for the genetic improvement of udder health and AFC in dairy animals.