Constitutional methylation of the MLH1 promoter is implicated in colorectal cancer (CRC) susceptibility by silencing the expression of the MLH1 protein. While MLH1 promoter methylation has been identified in variable frequencies among various populations, no data exist for CRC patients from Pakistan. In this study, we investigated constitutional MLH1 promoter methylation in Pakistani CRC patients. We screened 210 CRC patients belonging to HNPCC/suspected-HNPCC (n = 27) and non-HNPCC (n = 183) groups and 100 healthy controls for constitutional MLH1 promoter methylation using a methylation-sensitive high-resolution melting (MS-HRM) assay with methylated and unmethylated standards. Of the 210 CRC patients, 12.9 % (n = 27) had a family history of HNPCC-associated cancers, and 87.1 % (n = 183) had CRC with no family history (non-HNPCC group). The mean age at disease onset was 43.1 years (range 14–77), while controls had a mean age at enrollment of 40.0 years (range 19–74.4). Constitutional MLH1 promoter methylation was not identified in 210 CRC patients and 100 healthy controls. Constitutional MLH1 promoter methylation does not appear to be associated with CRC susceptibility in Pakistani patients.