Bovine leukemia virus (BLV) is a retrovirus that affects cattle worldwide. A longitudinal study was conducted with the aim to (a) estimate the incidence rate of the BLV infection of dairy farms in the regions of Los Ríos and Los Lagos (Chile), and (b) describe the frequency and epidemiological association of risk management practices related to new cases in cattle on dairy farms in Southern Chile. Infection status was based on commercial blocking ELISA results, on serum and milk. Individual information on animals and management practices was extracted from farm records, and then the most likely date of infection for new cases was estimated. The number of new infections was used to calculate the within-herd incidence rate. Adult animals had an incidence rate of 1.16 (95% CI 0.96; 1.20) cases per 100 cow-months at risk, while for young animals it was 0.64 (95% CI 0.44; 1.00) cases per 100 animal-months at risk. Rectal palpation, artificial insemination, and injections were the most common practices related to infection. Further studies are needed to determine if these are the only practices that facilitate spreading or if there are other practices that can be handled better in order to reduce the spread of BLV.