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To test the a priori hypothesis of HIV-1 transmission from one suspect to six recipients in a criminal case.Partial pol and/or env sequences were obtained for at least two samples of the suspect and the victims. Appropriate local controls were sampled based on epidemiological and subtype criteria. Phylogenetic testing was performed using different reconstruction methods.Phylogenetic analyses consistently inferred a monophyletic cluster for the suspect and victim samples in both genome regions. This was highly supported by parametric and non-parametric bootstrapping techniques. Moreover, the controls most closely related to the suspect–victim cluster had a similar geographical origin to the suspect.Taking into account the limitations on the conclusions that can be drawn from molecular investigations we could infer that our molecular data is consistent with a scenario of multiple HIV transmission between suspect and victims.