Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial-species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.