We examined the genetic associations of previously identified sequence variants with type 2 diabetes mellitus (T2DM) and its potentially genetic heterogeneity by gender in a large-scale cohort. A total of 613 T2DM patients and 8221 control subjects from the Korea Association REsource (KARE) cohort were included in the analysis of genetic association of T2DM with 33 nucleotide polymorphic markers identified by previous studies. The association analysis was further conducted with data partitioned by gender. The association analysis resulted in five nucleotide sequence variants associated with the susceptibility of T2DM after Bonferonni correction (P < 0.0015). One was located near the gene of hematopoietically expressed homeobox (HHEX), and the others were all in the gene of cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1). Further analysis revealed that the sequence variant (rs5015480) near HHEX and two SNPs (rs7756992 and rs9465871) in CDKAL1 were associated with the susceptibility of T2DM in females (P<0.005), but not in males (P>0.005). We suggested heterogeneous genetic associations of the T2DM susceptibility with the CDKAL1 and HHEX genes by gender.