ObjectivesTo identifyHaemophilus species and characterize antimicrobial susceptibility of isolates from patients with respiratory tract infections (RTIs) in Cameroon. MethodsIsolates (n = 95) were from patients with RTIs obtained from two Hospitals in Yaoundé, Cameroon. Isolates were identified by biochemical assay, PCR-based method, MALDI-TOF and whole genome sequencing. Antibiotic minimum inhibitory concentrations were determined by E-test. ResultsH. influenzae was the most prevalent species varying from 76.8% to 84.2% according to different methods. The isolates were mainly nontypable (n = 70, 96%). Three isolates of H. influenzae were capsulated (b, e and f). The isolates were genetically diverse and 40 unique sequence types were identified including 11 new ones. Resistance to ampicillin was observed among 55.3% (52/94) and 9% (14/52) produced TEM-1 β-lactamase. PBP3 mutations occurred in 57.7% of ampicillin resistant isolates (30/52). Eleven isolates were chloramphenicol resistant with 80% producing chloramphenicol acetyltransferase (8/10). Four Haemophilus isolates were rifampicin resistant with two mutations in rpoB gene. Five isolates were ciprofloxacin resistant and harbored mutations in the quinolone resistance determining regions of gyrA and parC genes. ConclusionH. influenzae isolates are highly diverse and show high levels of antibiotic resistance. H. influenzae serotype b is still circulating in the post-vaccination era.