We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma, Acholeplasma, Ureaplasma, Mesoplasma, and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes. This finding enabled the application of a combined polymerase chain reaction-microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.