We have assessed prevalence, incidence, and factors associated with increased risk for Chlamydia trachomatis genital infection among female migrant sex workers in Italy. In a prospective, observational study, women were offered free screening for sexually transmitted diseases and C. trachomatis was tested by a commercial ligase chain reaction assay in endocervical specimens. Of the 101 women tested, 14 (14%) were positive. The odds ratio (OR) for C. trachomatis infection was significantly higher for females under 24 years (OR=4.31), women from Eastern Europe (OR=4.80), and migrants less than 12 months in Italy (OR=4.41). In a multivariate logistic regression model, only origin from Eastern Europe remained independently associated to a higher risk for C. trachomatis infection (OR=6.05). This study provides evidence for high prevalence and incidence of C. trachomatis genital infection in migrant sex workers. Women from Eastern Europe have a significantly higher risk. These data reinforce the need for targeted control interventions.