Hygienic monitoring of laboratory rodents has focused more and more on the analysis of environmental sample material by quantitative polymerase chain reaction (qPCR) assays. This approach requires profound knowledge of specific genetic sequences of the agents to be monitored and the assays need to be permanently adapted to take the latest research into account. [Pasteurella] pneumotropica was recently reclassified into the new genus Rodentibacter, with Rodentibacter (R.) pneumotropicus and R. heylii as the most commonly detected species in laboratory mouse colonies. This study aimed at the development of a specific qPCR assay for the simultaneous detection of both agents. A novel primer probe set, based on detection of the specific virulence factor‚ ‘inclusion body protein A’ gene (ibpA), was confirmed by testing the assay on currently described Rodentibacter type species and other Pasteurellaceae. Furthermore, it was validated within four different barrier units and results were compared with the cultural analysis of sentinel mice. The assay was suitable to specifically detect R. pneumotropicus and R. heylii and discriminate them from other murine Rodentibacter spp. In addition, it revealed high sensitivity for the detection of both agents in environmental sampling material including exhaust air dust in individually ventilated cage systems. Altogether, higher pathogen prevalence was detected via qPCR of environmental samples compared with cultural diagnostics of sentinel mice. This study describes a qPCR assay for the simultaneous detection of R. pneumotropicus and R. heylii. This assay was demonstrated to be beneficial during routine health monitoring, especially with regard to environmental sampling strategies.