Chlamydia trachomatis, serovar L2, is the causative agent of lymphogranuloma venereum (LGV), which during recent years has been responsible for various outbreaks reported among men who have sex with men (MSM) in Western Europe, America, Canada and Australia. Samples from nine patients with chronic proctitis, seen at a local hospital were sent to us for identification of C. trachomatis serovar L2. The presence of C. trachomatis serovar L DNA was identified by realtime polymerase chain reaction (PCR) in two patients. They both had high positive C. trachomatis antibody titres (>/=10,000) and were found to be infected with serovar L2b by sequencing after amplification of the omp 1 gene by a nested PCR technique. These two individuals met the diagnostic criteria for LGV serovar L2b infection and, to our knowledge, these are the first cases described in Portugal.