BACKGROUND: Haemophilus ducreyi, traditionally recognized as the etiological agent of chancroid, a genital ulcer disease, is increasingly being identified as a significant cause of cutaneous ulcers in yaws-endemic regions across the South Pacific, Southeast Asia, and Sub-Saharan Africa. Despite its clinical relevance, this pathogen remains poorly characterized, and comprehensive genetic tools for analyzing isolate relationships are still lacking. METHODS: In this study, we present a follow-up of our previous research and developed a multilocus sequence typing (MLST) approach based on six of the seven loci from the Haemophilus influenzae MLST scheme and applied it to 82 primary clinical samples, previously confirmed to contain H. ducreyi, without culture. We also performed whole-genome sequencing (WGS) and antibiotic susceptibility testing on four cultured isolates obtained from cutaneous ulcers in yaws endemic health districts of Cameroon. RESULTS: Antibiotic susceptibility testing of H. ducreyi cultured isolates revealed sensitivity to all tested antibiotics, including ceftriaxone, azithromycin, and ciprofloxacin. MLST analysis, using data extracted from WGS and directly from clinical samples, identified 38 complete profiles across the six loci (34 from direct samples and four from cultured isolates), identifying 14 distinct sequence types (STs). BURST analysis of the six MLST genes grouped the STs into two distinct clonal complexes. An additional, polymorphism was observed in the ftsI gene, which encodes the penicillin-binding protein 3. CONCLUSIONS: This study highlights the need for genetic typing of H. ducreyi strains circulating in the yaws-endemic regions of Cameroon. The developed MLST scheme offered effective strain discrimination and provided valuable insights into their genetic relationships in these areas.