Objective Analysis of drug resistance mutations in the protease and reverse transcriptase gene and in the protease substrate cleavage sites of HIV-1 from AIDS pediatric plasma samples after long term treatment with protease and reverse transcriptase inhibitors for long time. Methods The protease, reverse transcriptase, gag and partial pol genes were amplified using Nest RT-PCR from 7 pediatric plasma samples. The PCR products of protease and reverse transcriptase gene were used directly for nucleotide sequencing. The PCR products of gag and pol gene were cloned into pCR2.1-TOPO TA cloning vector to be sequenced. The resulting nucleotide sequences were aligned and analyzed using Sequencing Analysis and HIV-1 Genotyping system software of PE company. Results Drug resistance mutations were found in the protease and reverse transcriptase genes from 6 patients' plasma samples. There are drug resistance mutations in the protease substrate cleavage sites in only 2 patients' plasma samples. Conclusions After long time(33～106 months) treatment of protease and reverse transcriptase inhibitors, drug resistance mutations in the protease and reverse transcriptase genes can be found in the most of the patients plasma samples(6/7), However, much less patients (2/7) were found to be with drug resistance mutations of the protease substrate cleavage sites.