Night shift work is associated with increased breast cancer risk, but the molecular mechanisms are not well-understood. The objective of this study was to explore the relationship between night shift work parameters (current status, duration/years, and intensity) and methylation in circadian genes as a potential mechanism underlying the carcinogenic effects of night shift work. A cross-sectional study was conducted among 74 female healthcare employees (n = 38 day workers, n = 36 night shift workers). The Illumina Infinium MethylationEPIC beadchip was applied to DNA extracted from blood samples to measure methylation using a candidate gene approach at 1150 CpG loci across 22 circadian genes. Linear regression models were used to examine the association between night shift work parameters and continuous methylation measurements (β-values) for each CpG site. The false-discovery rate (q = 0.2) was used to account for multiple comparisons. Compared to day workers, current night shift workers demonstrated hypermethylation in the 5ʹUTR region of CSNK1E (q = 0.15). Individuals that worked night shifts for ≥10 years exhibited hypomethylation in the gene body of NR1D1 (q = 0.08) compared to those that worked <10 years. Hypermethylation in the gene body of ARNTL was also apparent in those who worked ≥3 consecutive night shifts a week (q = 0.18). These findings suggest that night shift work is associated with differential methylation in core circadian genes, including CSNK1E, NR1D1 and ARNTL. Future, larger-scale studies with long-term follow-up and detailed night shift work assessment are needed to confirm and expand on these findings.