It is important to perform genetic quality control on inbred mouse strains to minimize variability of genetic backgrounds. We used sensitive molecular methods to examine the genetic integrity of inbred mouse substrains maintained at an academic institution. Our goal, in part, was to compare the different molecular genetic monitoring methods to determine which were most sensitive, efficient, and beneficial in our genetic monitoring program. We examined the sensitivity and efficiency of simple sequence length polymorphism (SSLP) analysis of microsatellites and restriction fragment length polymorphism (RFLP) analysis of minisatellites with commercial, human, and synthetic mouse minisatellite probes. Although no polymorphisms were detected with the microsatellite analysis, certain minisatellite probes detected a small degree of polymorphism between our mouse substrains and the commercially available strains used as controls. Minisatellite probes also detected intra-substrain variation within our colonies; this variation probably represents mutations in highly unstable loci rather than genetic variation. Our analysis indicated that the genetic integrity of in-house C57BL/Ka, BALB/cKa, and C3H/Km inbred substrains had remained intact over 35 generations. Genetic monitoring by RFLP minisatellite analysis was more sensitive and efficient in detecting substrain differences than was SSLP microsatellite analysis. On the basis of these results, we established a strategy for future analysis of the in-house breeding colony.