The purpose of this research study was to investigate the prevalence of the p.G61E variant of the CYP1B1 gene among primary congenital and open angle glaucoma (PCG and POAG) patients in the province of Punjab, Pakistan. A total of 112 POAG and 50 PCG patients were enrolled in this study. Detailed clinical examination was carried out on all patients. Screening of G61E was done by direct Sanger sequencing. Different in silico tools, e.g., Clustal Omega, PSIPRED, and Franklin tools were used to check the conservation, secondary structure, and pathogenicity, respectively, of this variant. Sanger sequencing of the whole CYP1B1 gene revealed a homozygous missense transition, c.182G>A, p.G61E in 25/50 (50%) of PCG and in 42/112 (37.5%) of POAG cases, which co-segregated with the disease phenotype. This study revealed that p.G61E is the relatively major contributor of PCG. However, 42 POAG patients harbouring the G61E mutation showed moderate to severe phenotype, suggesting the genetic heterogeneity of this variant in Pakistani population.