The same strain of rats purchased from different suppliers may exhibit behavioral differences that could give rise to variability and deficient repeatability. We have previously demonstrated that outbred Wistar rats from different suppliers display striking differences in voluntary alcohol intake. Using outbred male Wistar rats from five suppliers, four suppliers in Europe (Charles River, Taconic, Harlan and B&K Universal) and one in the United States (Harlan), the present study investigated the variability in behavioral profiles of the Wistar substrains. To this end, we used the multivariate concentric square field™ (MCSF) test. This test has an ethological foundation and is designed to provoke exploration and behaviors associated with risk assessment, risk taking and shelter seeking. The term “multivariate” refers to the test situation as well as the statistical analysis of the data. Our results demonstrate significant differences in body weight and behavioral profiles between the age-matched groups of Wistar rats. A principal component analysis clearly separated Harlan (US) and Charles River rats from the B&K Universal, Harlan (EU) and Taconic rats. The parameters important for this separation included activity and risk-taking performance. A trend analysis further confirmed this finding. The results emphasize the need for careful specification of the animals used in a given study. The utility of differences in explorative strategies and behavioral profiles among various outbred strains derived from a single strain is discussed. In conclusion, the data support differences between substrains of Wistar rats and illustrate the utility of using a multivariate strategy for behavioral profiling.