Background: To determine the prevalence of Haemophilus influenzae vulvovaginitis in prepubertal girls and the antimicrobial resistance of H.influenzae strains isolated from vulval specimens.Methods: Isolates of H.influenzae from vulval swabs of prepubertal girls with vulvovaginitis received at The Children's Hospital, Zhejiang University School of Medicine during 2016-2019 were studied. Vulval specimens were inoculated on Haemophilus selective chocolate agar. Antimicrobial susceptibility tests were performed using the disk diffusion method. A cefinase disk was used to detect β-lactamase. Results: A total of 4142 vulval specimens were received during the 4 years, 649 isolates of H. influenzae were isolated from 642 girls aged 6 months to 13 years, with a median of 5y. There were peaks of isolates from April to July seen in the vulval isolates. In total, the ampicillin resistance rate was 39.1% (250/640); 33.2% strains (211/636) were for β-lactamase-positive isolates, 6.6% strains (42/635) were β-lactamase-negative and ampicillin-resistant (BLNAR) isolates. The resistance rates of H. influenzae isolates to amoxycillin-clavulanic acid, ampicillin-sulbactam, cefuroxime, ceftriaxone, cefotaxime, meropenem, levofloxacin, sulfamethoxazole-trimethoprim, azithromycin, and chloramphenicol were 26.4%, 21.8%, 24.8%, 1.7%, 1.0%, 0.2%, 0%, 47.7%, 10.2%, and 1.1%, respectively. MDR was present in 41 (6.4%) of the 642 H. influenzae isolates, with the most prevalent MDR phenotype of ampicillin-sulfamethoxazole-trimethoprim-azithromycin resistance. Conclusions: H. influenzae is a common cause of vulvovaginitis in prepubertal girls. Laboratories should ensure that they include media appropriate for the isolation of H. influenzae. It’s worth noting of ampicillin resistance of H. influenzae in clinical management.