Background: The aim of this study was to analyze the microbiological characteristics of nasopharyngeal carriage Haemophilus influenzae isolates collected from children with respiratory infections in Beijing hospital and Youyang Hospital of China. Methods: The serotypes of all isolates were determined using latex agglutinated antisera (a-f). The minimum inhibitory concentrations (MICs) of 11 antibiotics were determined using E-test strips. For the beta-lactamase-negative ampicillin-resistant (BLNAR) isolates, ftsI gene was sequenced based on fragments amplified by PCR. STs of H influenzae isolates were determined by multi-locus sequence typing. Results: The overall carriage rate of H influenzae in the study population was 9.1% (362/3984). One hundred and ninety H influenzae isolates which were selected in our study were non-typeable (NTHi) and 44 (23.2%) of them were positive for β-lactamase. All isolates were susceptible to ceftriaxone and levofloxacin. Susceptibility rates to erythromycin and sulfamethoxazole-trimethoprim in Beijing were significantly higher than Youyang (P <.05). Thirty-six BLNAR isolates were identified. The MLST analysis showed 108 STs in 190 isolates, the most common of which were ST408 (11, 5.8%), ST914 (10, 5.3%), ST57 (9, 4.7%), and ST834 (6, 3.2%). Twelve STs were detected in both of the study sites, which covered 63 isolates. Conclusions: All isolates in the present study were NTHi, which suggested widespread of this type in China. The BLNAR isolates were detected more frequently than before. Because high genetic diversity of NTHi isolates of H influenzae exists worldwide, it is important to continuously monitor these bacteria in the future. © 2019 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.