Abstract Background In recent years a few cases of lymphogranuloma venereum (LGV) in heterosexuals in Europe have been reported. It is not known whether LGV transmission among heterosexuals occurs on a wider scale. Methods Heterosexual male and female STI clinic clients (n = 587) in Amsterdam, the Netherlands, with a positive nucleic acid amplification test (NAAT) result for Chlamydia trachomatis (CT) were screened for IgA anti-MOMP in serum. If the value was above the cut-off index (2.0) the patient’s CT positive urogenital, ocular or rectal sample(s) were selected and tested for LGV by an in-house LGV-specific NAAT. Results Sera of 126 patients were above 2.0 COI. Some patients had >1 CT positive sample. Samples could not be retrieved from 15 of the 126 persons, and 7 samples that were found positive for CT in the diagnostic amplification process could not be confirmed and hence not typed. We did not find a single case of LGV infection in 123 urogenital, ocular or rectal samples from 104 patients. Conclusion We found no indications for significant spread of LGV infection in heterosexuals in Amsterdam. Surveillance in females with cervical or anal CT infection is indicated to monitor LGV occurrence in heterosexuals.