Lysozyme, an anti-bacterial enzyme, is mostly found in the body fluids, various tissues and secretions of animals and humans, and confers immunity against a wide range of bacterial species. The present study was carried out to elucidate the gene sequence of this enzyme in Indian Sahiwal × Holstein Friesian crossbred cattle and to explore the polymorphism of the gene as well as their association with milk production and somatic cell traits. The total length of lysozyme cDNA was found to be of 447 bp. The similarity with Bos taurus, human, pig, monkey, gorilla, mice, rat, chicken, dog and sheep was estimated as 99.1%, 85%, 81.0%, 85.2%, 84.3%, 77.9%, 77.9%, 41.4%, 40.5% and 24.6%, respectively. Polymorphism study of two fragments, extended exon1 including promoter, exon1 and partial intron1 (268 bp), and extended exon2 including partial intron1 and 2, and exon2 region (287 bp) of milk lysozyme gene was carried out by employing single-stranded conformation polymorphism (SSCP). In the extended exon1 fragment, three alleles namely A, B and C with frequencies of 0.59, 0.28 and 0.12 were observed while in the exon extended fragment, three different alleles – P, Q and R with respective frequencies of 0.61, 0.38 and 0.01 – were determined in Indian Sahiwal × Holstein Friesian cross. A total of eight haplotypes were found in this population where the most predominant one was h1 (0.52). Genotypes of exon1 extended fragment showed significant association with total milk yield, daily milk yield, peak yield and somatic cell score at P < 0.05 while that of exon2 extended fragment had significant correlation with only total lactational milk yield. Haplotype combinations also revealed significant association with total milk production where h1h1 homozygous showed highest yield during first lactation.