Internal transcribed spacers in ribosomal DNA were often used as molecular sequence in phylogenetic study at the population and species level.PCR was used to isolate the complete sequence of internal transcribed spacer(ITS1-5.8S-ITS2) and partial 18S and 28S sequence from Oreochromis aureus and O.niloticus.The analysis of ten clones from four individuals indicated that O.aureus had two types of ITS1(ITS1a and ITS1b) with different lengths.ITS1a was 536 bp,GC content was 69.96%;ITS1b was 520 bp,GC content was 69.04%-69.42%.The analysis of ten clones from four O.niloticus individuals indicated that only ITS1a existed in O.niloticus which was 536-540 bp in length,and 69.42%-70.19% in GC content.Compared with ITS1b,ITS1a had 16 bp sequences(GGCCCGCCTGCGGCGC) which was inserted from 16 nt to 31 nt.5.8S of all the 20 sequences from O.aureus and O.niloticus was 157 bp,GC content was 56.69%-57.96%,and ITS2 was 408 bp,GC content was 72.79%-74.26%.The high identity of ITS between O.aureus and O.niloticus indicated that the genetic relationship of the two Oreochromis was very close.Furthermore,PAGE electrophoresis was used to investigate the ITS1 divergence of O.aureus,O.niloticus and O.aureus(♂) ×O.niloticus(♀) with distinguished morphological characters.The analysis showed that all of the 14 O.aureus individuals had two types of ITS1;1 individual of the 15 O.niloticus and 6 individuals of the 15 O.aureus(♂) ×O.niloticus(♀) had two types of ITS1.This paper showed the population of O.aureus had high identity while 1 individual of O.niloticus was genetic mixture with O.aureus in the locus of ITS1 and it also indicated that the means of molecular biology is more accurate than the means of morphology in germplasm identification.