The objective of this study was to assess the patterns of genotypic and phenotypic resistance in a population of blood donor patients infected with HIV-1 subtype B' (Thai B', a clade of HIV-1 B) from central China, previously treated and harboring NRTI and NNRTI resistance mutations, with the purpose of designing effective therapeutic regimens. The HIV-1 pol genes from 65 patients were sequenced and estimated for drug resistance while the viruses isolated from the patients were used to analyze the phenotype based on the TZM-bl cell line. All the HIV-1 strains harboring one or more drug resistance mutations to HIV-1 RTIs possessed high cross-resistance to EFV (100%) and DLV (92%), as well as to ABC (84%) and TDF (77%), which are much higher than both FTC and 3TC (42%). There were more thymidine analog mutation (TAM)-associated mutations in the AZT/ddI/NVP group (62.5%) than in the d4T/ddI/NVP group (32.65%). A phenotypic assay showed high concordance between genotypic and phenotypic cross-resistance. This study showed there was a high level of cross-drug resistance to HIV-1 RTIs among Chinese AIDS patients harboring resistant strains, and there is also a high prevalence of primary resistance to 3TC, suggesting that one important recommendation should be the realization of genotypes in all naive patients due to the high prevalence of NRTI and NNRTI mutations.