Eighteen reference strains of Chlamydia trachomatis were differentiated by omp1 PCR- and nested PCR-based RFLP analysis, using two restriction digestions, one with AluI and the other with the three enzymes HpaII, EcoRI and HinfI. AluI digestion allowed the differentiation of 12 different profiles after CT1/CT5 PCR and 13 different profiles after the nested PCR. The triple hydrolysis permitted the identification of 15 different patterns. In all, 16/18 reference strains were clearly identified. These reference patterns were successfully used to genotype 34 of 35 (28 strains and 7 clinical specimens) samples from infected students, collected during a screening programme in Yaounde (Cameroon). Genotypes D, Da, E, F, G and J were found. The most prevalent omp1 genotype was E (n = 14; 40 %), followed by F (n = 7; 20 %). As RFLP patterns of reference strains are essential for typing clinical isolates, they will greatly facilitate C. trachomatis characterization in many resource-limited laboratories.