Objective Haemophilus influenzae type b (Hib) is the most virulent serotype that colonizes the upper respiratory tract of children. After introducing Hib conjugate vaccine, Hib has become almost entirely replaced by nontypeable H. influenzae (NTHi) strains. This study investigated the incidence of NTHi, which may cause serious sporadic body infections. Methods A total of 300 nasal and throat swab samples were obtained from children aged 6 to 60 months, who visited outpatient pediatric clinics of Jordan University Hospital and Al-Bashir Hospital, Amman, Jordan. Colonies resembling H. influenzae were tested by conventional laboratory tests including Gram staining and antibiotic susceptibility. The 16S rRNA and bexA (capsulation gene) were amplified from the extracted DNA. Swabs (n = 100) were delivered to the laboratory in transport media. After initial culture, DNA was extracted directly and tested by conventional polymerase chain reaction (PCR) employing actin beta primers for assessing the housekeeping gene, followed by quantitative PCR (qPCR) against Hib-specific primers. Results A total of 31/300 (10.33%) of H. influenzae isolates were recovered by culture and confirmed by PCR for 16S rRNA; 96.77% (30/31) were NTHi, and only one isolate was Hib. This isolate was confirmed by detection region II of the cap locus of Hib gene by qualitative TaqMan real-time qPCR. Antibiotic resistance among NTHi to ampicillin and erythromycin was 50.0 and 20.0%, respectively. Conclusion The conjugated Hib vaccine almost eliminated the occurrence of Hib in the upper respiratory tract of the Jordanian children, while NTHi colonization was found in 10% of them.