Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.