Canine leptospirosis has been described as having re-emerged in North America around the mid-1990s, with a change in the epidemiology of the infecting serovars responsible for the disease emergence. A retrospective case-control study was conducted to examine the re-emergence of seroprevalent cases of canine leptospirosis in Ontario using serology submission records from 1406 dogs from January 1, 1998 to December 31, 2006. The data collected [results of the microscopic agglutination test (MAT), veterinary clinic postal code, age, sex, neutering status, and breed] were analyzed by multivariable logistic regression, generalized linear mixed modeling, and Cochran-Armitage test for trends in proportions. Dogs in urban areas appeared to be at significantly higher risk than dogs in rural areas for the entire study period [odds ratio (OR) = 1.6, confidence interval (CI) = 1.2–2.3], though this was not as marked as in other studies. Results indicated that canine leptospirosis in Ontario is a disease of all breeds and ages, regardless of gender. No geographic clustering was noted, but clustering of cases by clinic within geographic areas suggested differences in awareness or in diagnosis by veterinarians. A distinctive seasonal pattern of leptospirosis, with more cases occurring during the summer and fall, as found in previous studies, was also observed in this study. The temporal trend analysis was consistent with an increasing proportion or re-emergence of seroprevalent cases of canine leptospirosis since 1998, suggesting that the putative increase in canine leptospirosis has been genuine.