The SPF10-PCR-based line probe assay (LiPA-25) for human papillomavirus (HPV) genotyping with high analytical sensitivity and specificity was widely used in HPV vaccine clinical trials and epidemiologic studies. In the study, we aimed to compare a novel GP5+/6+-PCR-based reverse dot blot hybridization assay (Yaneng-23) with LiPA-25. The performance of two assays was evaluated in 1735 cervical swab and 117 tissue samples, with a focus on 19 common HPV types (14 high-risk: 16,18,31,33,35,39,45,51,52,56,58,59,66 and 68/73; 5 low-risk: 6,11,42,43 and 53). A total of 1197 (69.0%) swab samples were identified as HPV-positive by two assays. Of these, 878 (73.4%) samples displayed absolute agreement (concordant), 255 (21.3%) showed additional or fewer types (compatible), and the remaining 64 (5.3%) samples were discordant. Additionally, the two assays showed an excellent strength of agreement for 19 HPV-combined detection (κ=0.886) and 17 individual HPV types (all κ>0.800), and displayed a good agreement for HPV39 (κ=0.780) and 42 (κ=0.699). Yaneng-23 was more sensitive than LiPA-25 for HPV58, 59, 68/73, 42, 43 and 53 (McNemar's test: all P<0.05), while LiPA-25 was more sensitive for HPV31, 39, 52 and 66 than Yaneng-23 (all P<0.05). In 113 HPV-positive tissue specimens, the identification of genotypes was 82.3% identical and 17.7% compatible. The agreement between the tests for HPV45 (κ=0.796) and 51 (κ=0.742) was good, and for other types (all κ>0.843) and 19 HPV-combined detection (κ=0.929) was perfect (all P>0.05). In conclusion, Yaneng-23 and LiPA-25 are comparable. Yaneng-23 could be used for the detection and genotyping of HPV in cervical samples for epidemiological and vaccine studies worldwide. This article is protected by copyright. All rights reserved.