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Objectives: We aimed to utilize a simple molecular assay to simultaneously detect both group B Streptococcus (GBS) and virulent ST-17 rectovaginal colonization. We also attempted to estimate the prevalence of maternal GBS and ST-17 carriers and to evaluate their seasonal association. Subjects and Methods: We used an optimized multiplex PCR method employing scp-B and ST-17 primers to analyze DNA extracted from rectovaginal swabs of 3,064 cases collected over 3 years. The incidence trends, seasonal variations, and temperature preference were analyzed. Results: The overall prevalence of maternal colonization for GBS and ST-17 clone were 13.25 and 2.48%, respectively. The ST-17 to GBS ratio was 18.72%. The occurrence of ST-17 colonization was significantly associated with seasonal variations with a preference for lower temperatures. Conclusions: We developed a novel multiplex PCR method suitable for the simultaneous detection of GBS and ST-17 clone. The phenomenon of lower temperature preference for ST-17 clone necessitates further investigation. The epidemiological data for GBS and ST-17 incidence are especially important to establish a public policy for universal GBS screening in the future.