Abstract: Objective: To compare next-generation sequencing（NGS） and third-generation sequencing（TGS） technologies in HIV-1 subtype determination, drug resistance mutation detection, and identification of high-and low-frequency variants. Methods: A total of 50 newly reported HIV-1-infected cases from Guangxi, China were enrolled. Near full-length genomes were sequenced using both NGS and TGS platforms. Subtype classification, resistance-associated mutations, and mutation frequency distributions were analyzed using appropriate online platforms and software. The inconsistent results of subtypes and drug resistance between NGS and TGS were verified by Sanger sequencing. Results: The two sequencing technologies showed good concordance in subtype determination（Cohen's Kappa=0.900, P<0.001）. Among the samples, five were classified as indeterminate by both NGS and TGS, and two showed discordant subtype assignments, primarily involving low-prevalence or recombinant subtypes. TGS identified one additional NNRTI resistance-associated mutation（V179E and V106I） and showed superior performance in detecting complex drug-resistant mutation patterns. There was a difference in the distribution of cumulative variant sites across different mutation frequencies identified by the two technologies（χ²= 3,771.87, P<0.001）. NGS cumulatively detected 18,367 variant sites（frequency≥5%）, with ultra-high-frequency mutations（≥80%） predominating 84.1%. In contrast, TGS cumulatively identified 28,120 variant sites with a frequency ≥5%, of which ultra-high-frequency mutations accounted for 58.1%. Within the mutation frequency range of 5%-60%, TGS detected a greater number of variant sites than NGS. The overall distributions of cumulative mutation site counts at different frequencies detected by the two sequencing technologies were significantly different in samples with concordant subtype classification and in samples with discordant or ambiguous subtype assignments（χ²=3,106.93, P<0.001; χ²=717.26, P<0.001）. Conclusion: NGS and TGS yield consistent results in HIV-1 subtype classification. TGS offers higher sensitivity for complex and low-frequency mutations, while NGS is more stable for ultra-high-frequency variants.