BackgroundNon-typeableHaemophilus influenzae(NTHi) has become the major cause of invasiveH. influenzaediseases in the post-H. influenzaetype b vaccine era. The emergence of multidrug-resistant (MDR) NTHi is a growing public health problem. Herein, we investigated the molecular basis of MDR in NTHi. The isolated NTHi were subjected to antimicrobial susceptibility testing for 12 agents. Whole genome and plasmid sequencing were conducted and analyzed to identify significant genetic variations and plasmid-encoded genes conferred antibiotic resistance.ResultsThirteen (50%) MDR NTHi isolates were obtained; of these, 92.3% were non-susceptible to ampicillin, 30.8% to amoxicillin-clavulanate, 61.5% to cefuroxime, 61.5% to ciprofloxacin/levofloxacin, 92.3% to trimethoprim-sulfamethoxazole, 30.8% to tetracycline, and 7.7% to azithromycin. Eight ampicillin-resistant isolates were β-lactamase positive; of these, 6 carriedblaTEM-1and 2 carriedblaROB-1, whereas 4 were β-lactamase negative. Genetic variations inmrdA,mepA, andpbpGwere correlated with amoxicillin-clavulanate non-susceptibility, whereas variations inftsIandlpoAconferred cefuroxime resistance. Five variations ingyrA, 2 ingyrB, 3 inparC, 1 inparE, and 1 in theparC-parEintergenic region were associated with levofloxacin/ciprofloxacin non-susceptibility. Among these genes, 8 variations were linked to high-level levofloxacin resistance. Six variations infolAwere associated with trimethoprim-sulfamethoxazole resistance. Plasmid-bearingtet(B)andmef(A)genes were responsible for tetracycline and azithromycin resistance in 4 and 1 MDR isolates, respectively.ConclusionsThis study clarified the molecular epidemiology of MDR in NTHi. This can benefit the monitoring of drug resistance trends in NTHi and the adequate medical management of patients with NTHi infection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02926-6.