To date there have been few reports of a gender difference in methylation levels of genes. When examining the methylation levels of four autosomal genes (ESR1, MTHFR, CALCA and MGMT) in the white blood cells of a random sample of Singapore Chinese Health Study cohort participants (n = 291), we encountered an unexpected gender differential. Using MethyLight technology, we calculated a gene-specific percentage of methylated reference (PMR) value, which quantified the relative level of gene methylation for each study subject (134 males and 157 females). Two summary methylation indices were constructed by assigning gene-specific rank scores. We then used ANCOVA to compare logarithmically transformed individual PMR values and summary methylation indices by age and gender simultaneously. Adjustment was made for plasma homocysteine. For ESR1, for which a large proportion of subjects were negative for methylation, we also used polytomous regression to compare methylation across age and gender. Increasing age and the male gender independently predicted increasing PMR values for CALCA and MGMT. For the MTHFR gene, male gender was associated with higher PMR values (P = 0.002), while age was not (P = 0.75). Neither age nor gender had any statistically significant influence on the PMR values for ESR1 (P = 0.13 and 0.96, respectively). Our data suggest that gender is at least as strong a predictor of methylation level in the four genes under study as age, with males showing higher PMRs.