Stenotrophomonas maltophilia is an emerging opportunistic pathogen linked not only to bacteremia, sepsis, and pneumonia but also to severe chronic enteritis. Persons with the impaired immune system are prone to be infected by S. maltophilia since its pathogenicity seems to be more associated with the host immune system than with the acquisition of specific virulence genes. In the dairy chain, S. maltophilia is linked to clinical and subclinical bovine mastitis in dairy cows, and it has been identified in cheese, and raw and pasteurized milk. There are reports of misidentification of S. maltophilia by commercial systems and PCR assays using primers based on the 23S rRNA and smeD genes, so the smeT gene is an alternative to identifying S. maltophilia by PCR due to its specificity to the S. maltophilia species. The present study reports an alternative species-specific PCR assay based on the smeT gene designed to identify S. maltophilia in cheese samples. We performed in silico and in vitro analyses to check the specificity of the primer pair. In silico analysis showed specificity of the primer pair to the species level. In vitro analysis was performed by testing the primer pair against pools of bacteria grown from 33 fresh Minas cheese samples acquired in the city of Rio de Janeiro, Brazil, without unspecific amplification.