Objective: The utility of capsular typing by nested PCR method was evaluated. The prevalence of Haemophilus influenzae type b (Hib) strains among ampicillin-resistant isolates was determined. Methods: A total of 899 H. influenzae strains isolated from nasopharyngeal specimens of children with respiratory tract infection were investigated in Beijing, Shanghai and Guangzhou during the past four years from 2000 to 2003. Susceptibility to ampicillin was determined by E test. Nested PCR with primers specific to bexA gene and capsular type-specific genes was established. Hib strains among resistant isolates were identified by nested-PCR based capsular typing and slide agglutination serotyping (SATS) at the same time. Results: Of the 74 H. influenzae strains resistant to ampicillin, 2 were positive by nested-PCR, and characterized as genotype b. The prevalence of Hib in ampicillin-resistant Hi isolates was about 2.7%. Conclusions: Nested-PCR based capsular typing as a supplement to slide agglutination serotyping, is useful for identifying Hib. The prevalence of type b strain among ampicillin-resistant H. influenzae isolated from nasopharyngeal specimens of children with respiratory tract infection in a defined geographic area during the past four years is at relatively lower level.