The present invention encompasses nucleic acids, methods, compositions, and kits for sensitive, rapid and specific detection of Mycoplasma, Acholeplasma, Ureaplasma, Phytoplasma and Spiroplasma species in a sample. The invention utilizes specific primers and amplification methods that permit differentiation between species due to specific amplification of target nucleic acids of contaminating Mollicute cells. In one embodiment, the invention utilizes the differing melting temperatures (Tm) of various potential PCR products to identify whether they are specific target amplification products, non-specific, non-target amplification products, specific positive control products, or primer-dimer products.