The need to take sex into account in biomedical research is now recognized and mandated by funding institutions. In laboratory rodents, such as mice, sexing is usually performed anatomically or by genotyping using multiplex or simplex PCR techniques on genomic DNA. Here we present a simple RT-PCR-based method targeting Kdm5c and Kdm5d to determine genetic sex in mouse cDNA samples, allowing for retrospective sex determination.