BACKGROUND: Human rhinoviruses (HRVs) are frequent etiologic agents of tract infections, ranging from benign upper to potentially life-threatening lower respiratory tract infections. Diagnosis is based on molecular methods. 169 HRV types, belonging to species A, B and C, have been identified. This high genetic diversity makes it difficult to accurately detect circulating HRVs and to diagnose severe infection. OBJECTIVES: To comparatively assess the ability to detect HRV clinical isolates of the first version (V1) of the commercial real-time RT-PCR Rhino&EV/Cc r-gene((R)) (bioMerieux) kit, of an in-house RT-PCR followed by genotyping, considered as the reference method, and of the second version of this commercial test (V2). STUDY DESIGN: From September 2011 to April 2013, HRVs were prospectively detected in 2525 respiratory specimens, using V1 in combination with the in-house reference RT-PCR. In November 2013, 85 specimens that had given initially false negative results with V1 were retested simultaneously with V1 and V2 and the in-house RT-PCR. In addition, 421 negative specimens with the in-house assay were prospectively tested with V2. RESULTS: Among the 2525 specimens, V1 detected 80.7% (502/622) of in-house RT-PCR positive isolates: 85.3% (220/258) of HRV-A, 84.4% (27/32) of HRV-B and 74.9% (176/235) of HRV-C. Among the 85 respiratory samples tested with V1, V2 and the in-house RT-PCR, V2 was more efficient than V1 in detecting 16 HRV isolates: 11/33 (33.3%) of HRV-A and 5/47 (10.6%) of HRV-C tested. The analytical sensitivity of V2 was greater for 8/18 HRV-A genotypes and 2/22 HRV-C genotypes. Relative to the in-house assay, the specificity of V2 was 100% (421/421). CONCLUSIONS: This study showed a slightly higher sensitivity of V2. However, diverse genotypes, especially HRV-C, were undetected.