Background: This study aims to compare cytomegalovirus (CMV) DNA detection rates in reproductive tract secretions (RTS) and urine samples and to further evaluate whether multiple specimen testing improves overall CMV DNA detection rates. Methods: We conducted a retrospective cohort study involving 3,804 female participants who provided 4,383 samples (10.95% RTS; 89.05% urine) between January 2020 and June 2024. Analyses included: (1) CMV DNA detection rates across sample types and population subgroups, (2) the impact of single versus multiple specimen testing on CMV DNA detection rates Result: CMV DNA was detected in 3.03% (133/4383) of all samples. Positivity rates differed significantly: 9.79% (47/480) for RTS versus 2.20% (86/3903) for urine ( p < 0.001). Single-sample testing yielded a positivity rate of 2.46% (82/3,330), while testing multiple specimens increased this to 5.91% (28/474). Concurrent testing of both urine and RTS yielded the highest positivity rate (15.63%, 20/128). Additionally, median CMV viral load was significantly higher in RTS compared to urine (3.34 ± 1.43 vs . 0.81 ± 1.47, p < 0.001). Conclusions: The detection rate of CMV DNA in RTS is higher than that in urine. The combined sample testing increases the detection rate of CMV DNA, potentially helping identify women with active viral shedding. These findings inform the development of optimized CMV DNA testing protocols.