This paper tries to compare the diversities of endophytic bacteria from Bt transgenic cotton and its parent cotton, and provides basic data for the biosafety evaluation of Bt transgenic cotton. Endophytic bacteria were isolated separately from the stems of two cottons (Zhongmian 30, Bt transgenic cotton, TC; Zhongmian 16, contrast non-transgenic parent, CC) by culture-dependent method. 16S rDNAs were amplified with universal primers of bacteria, and PCR products were digested with restriction endoenzyme Hha I and Rsa I respectively, than analyzed by RFLP analysis. The results showed that endophytic bacteria content was 105 cfu/g. There were 10 OTUs (operational taxonomic units) in the stems of Bt transgenic cotton and 9 OTUs in contrast parent cotton. The 16S rDNA sequences of 12 respective strains were identified and submitted to GenBank. 16S rDNA sequencing indicated that the dominant genera of Bt transgenic cotton are Microbacterium and Curtobacterium. The predominance families of corresponding receptor cotton are Curtobacterium and Enterobacter. Bt transgenic cotton did not bring harmful effects upon the number and diversity of endophytic bacteria in stems.