Genetic variability of the nontranscribed spacer (NTS) region within ribosomal DNA repeating units in the various subspecies of Mus musculus was determined. Mice belonging to several laboratory mouse strains were examined by means of Southern blot hybridization with a mouse ribosomal DNA probe. This probe encompasses the 3' end of the 28S ribosomal RNA (rRNA) gene and the following spacer. Restriction enzyme digestions of the liver DNAs from various wild mice revealed that each of the subspecies has a unique pattern in the spacer encompassing a distance approximately 10 kb downstream from the ribosomal gene. These restriction patterns permit the classification of mouse subspecies and also provide insights into the origin of the laboratory mouse strains.