Leptospirosis is a ubiquitous distributed zoonotic infectious disease present in wild and domestic animals. This study aimed to estimate within-herd and herd-level seroprevalence against pathogenic Leptospira spp. in dairy cows from southern Chile and identify risk factors associated with the herd-level status. We used a multi-stage strategy combined with a stratified sample strategy for randomly sampling 147 herds and 4876 lactating cows. We considered as infected a herd with at least one positive reactor to MAT. In addition, an epidemiological survey was applied to the herd’s owners and a logistic regression (LR) model was constructed to analyze it. The overall within-herd prevalence was 5.9% (95% CI 4.9–6.8), the overall herd-level prevalence was 42.2% (95% CI 9.2–47.9), and there was variation in both between different herd sizes. L. borgpetersenii serovars Hardjo and Tarassovi and L. interrogans serovar Pomona were the more frequent serovars in non-vaccinated herds. A factor that decreases the risk of a farm being infected was Leptospira vaccine usage (OR = 0.04; 95% CI = 0.02–0.11), and variables that increase risk were using bulls for mating (OR = 3.43; 95% CI = 1.1–10.1) and continuous calving distribution (OR = 3.4; 95% CI = 1.3–8.8). The study’s results will contribute to unravelling the infection burden in the main dairy area of the country and designing control strategies.