Lactose non-persistence (LNP) develops due to the downregulation of the lactase-phlorizin hydrolase (LPH) enzyme. LPH is encoded by the LCT-gene located on chromosome 2q21. In this study, 50 patients with LI were investigated for the identification of mutations in the LCT-gene. In comparison of 30 subject with lactose persistence (LP) considered as healthy group. A total of 13 genomic mutations were identified, 1 in the promoter region and 12 in the intronic/exonic region of LCT-gene. Among these 12 mutations, 6 of them are novel in origin. The novel mutations were found in intron 4 c.(918+116)A>C, intron 12 c.(4877+20)G>A, intron 15 c.(5346+35)T>C, UTR of exon 17 (c.5865C>T), exon 6 c.1147A>G and c. 1095A>G. Four mutations resulted in an altered amino acid sequence. The frequency of each mutation observed in both gruops was: mutation I of exon 1 (0.64/1), mutation II of exon 2 (0.34/0.67), mutation III, IV and V of exon 6 (1/1,0.12/0,0.64/1) mutation VI of exon 13 (0.64/1), mutation VII, VIII, IX of exon 17 (0.64/1, 1/1, 0.12/0), mutation X of intron 4 (0/1), mutation XI of intron 12 (1/0), mutation XII of intron 15 (1/1) and mutation XIII of promoter region (0.36/0.67). In this study, molecular identification and screening in LNP subjects provide the mutational variants of the LCT-gene in the region of Pakistan. This is a major step in clinical management and accurate genetic counseling of the pre-symptomatic diagnosis of LNP. Among the six novel mutations found, mutation XI was found in all of the LNP subjects and was absent in the lactose persistent group. This study, for the first time, focuses on molecular analysis of LCT-gene from Pakistani patients with LNP.