Objective: The objective was to study the association of Klotho gene G395A and C1818T single nucleotide polymorphisms with glycemia, serum, glycosylated hemoglobin (HbA1c) level and the risk of type 2 diabetes mellitus (T2DM) in the Pashtun population of Pakistan. Methods: In this study, 195 normal individuals and 217 T2DM patients were enrolled. All subjects were divided into three groups, namely overall subjects (control + T2DM patients), control individuals and T2DM patients, and their fasting glucose, HbA1c level, lipid profile and C1818T and G395A polymorphisms were determined. Results: The allele frequencies of G395A in overall subjects were 0.568 for A and 0.432 for G. Similarly, allele frequencies for G395A in overall subjects were 0.597 and 0.403 for C and T alleles, respectively. The AA genotype of G395A was observed to be a risk factor for T2DM. In normal individuals, no significant (p > 0.05) association was observed between klotho C1818T and G395A polymorphisms and hyperglycemia. In overall subjects, the C1818T polymorphism was associated (p < 0.05) with high fasting glucose and HbA1c levels in female subjects only. In T2DM patients, both C1818T and G395A polymorphisms were found to be significantly (p < 0.05) associated with high fasting glucose and HbA1c levels both in males and females. Conclusion: The G395A polymorphism was observed to increase the risk of T2DM. Both C1818T and G395 were associated with high fasting glucose and HbA1c levels in T2DM patients.