Background: Chlamydia trachomatis is a common sexually transmitted infection in Chile, but little is known about the genovar distribution in genital infections. Thus, the objective of this study was to determine the distribution of C. trachomatis genovars in such cases. Methods: A total of 522 urogenital specimens, 403 from women and 119 from men, were analyzed for C. trachomatis by nested polymerase chain reaction (PCR) targeting of the ompA gene. Positive specimens were genotyped by DNA sequencing of the amplicons. Results: Sixty-two (11.9%) specimens were positive. Of these, 43 (69.4%) were collected from men and 19 (30.6%) from women (p < 0.0001). Eight genovars were identified in men and seven in women. Genovar E was the most common in both men and women, followed by genovar Da in men, and F in women. Together these three genovars accounted for 84% of infections. Genovar D was the third most common genovar (n = 4). Genovar G was detected in two samples, and sequences of genovars Ba, H, and Ja were each found in single samples. One sample (1.6%) contained mixed sequences. No association was found between gender and specific genovars. Fifty-six (92%) sequences were identical to those reported for the respective reference genovars and the other two have been described in several regions. Conclusions: Our findings add to the results of most studies, which indicate that genovars E, F, and D/Da are the most frequent. No association was found between gender and specific genovars. Despite the heterogeneous population of genovars, most ompA sequences were conserved.