Aims To perform a systematic evaluation of the applicability, validity and reliability of the long PCR-RFLP of 16S-ITS-23S rRNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures. Methods and Results 3301 Full-length sequences of 16S-ITS-23S rRNA genes were retrieved from 885 published bacterial genomes. Copy numbers of the whole set of 16S-ITS-23S rRNA genes per genome ranged from 1 (n = 161) to 14 (n = 4) with an average of 3.71. Their length varied greatly, from 4319 to 6568 bp with an average of 4952 bp. Computer-simulated RFLP analyses of the 16S-ITS-23S fragments flanked by the conserved primers 27F and 2241R suggested MspI, RsaI, HhaI and TaqI as the most appropriate enzymes for long PCR-RFLP analysis of the 16S-ITS-23S sequence. MspI was used to screen over 900 bacterial cultures isolated from the Huguangyan Maar Lake in southern China. An experimental sequencing of 16S rRNA genes of the isolates possessing a unique RFLP band pattern proved the broad applicability and high resolution of this approach. Conclusions These results indicate that long PCR-RFLP of 16S-ITS-23S rRNA genes is a potentially universal and reliable bacterial genotyping tool with a high resolution. Significance and Impact of the Study The methodology of long PCR-RFLP of 16S-ITS-23S rRNA genes will facilitate the exploration and tracing of cultivable microbial diversity in natural environments.