The SPF10-PCR-based line probe assay (LiPA-25) with high analytical sensitivity and specificity for HPV genotyping in clinical samples has been widely used in vaccine and epidemiologic studies. A real-time multiplex PCR assay using type-specific primers (Hybribio-23) with low workload and cost has been developed recently. The study aimed to compare the performance of LiPA-25 and Hybribio-23 in selected 1731 cervical swab and 117 tissue samples, with a focus on 20 common HPV types (14 high-risk: 16,18,31,33,35,39,45,51,52,56,58,59,66 and 68/73; 6 low-risk: 6,11,42,43,44 and 53). The level of agreement of two assays was determined using Cohen's Kappa (κ) statistics. A total of 1296 (74.9%) swab samples were identified as HPV-positive by Hybribio-23 or LiPA-25, of which 814 (62.8%) samples exhibited concordant, 358 (27.6%) showed additional or fewer types (compatible), and 124 (9.6%) were discordant. In addition, the two assays showed a perfect agreement for 20 HPV-combined detection (κ=0.838) and 17 individual HPV types (all κ>0.800), a good agreement for HPV31 (κ=0.792) and 43 (κ=0.696), and a moderate agreement for HPV42 (κ=0.504). Hybribio-23 was significantly more sensitive for HPV58, 59, 68/73, 42, 43 and 44, and less sensitive for HPV35 and 66 than LiPA-25 (McNemar's test: all P<0.05). For 117 HPV-positive tissue specimens, the identification of genotypes was 85.2% identical, 12.2% compatible, and only 2.6% discordant. The agreement for HPV31 (κ=0.786), 68/73 (κ=0.742) and HPV53 (κ=0.742) was good, while for other types (all κ>0.853) and 20 HPV-combined detection (κ=0.936) was perfect (all P>0.05). In conclusion, Hybribio-23 and LiPA-25 are comparable. Hybribio-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.