A cross-sectional serological survey was conducted during January to August 2001 to determine the seroprevalence of Leptospira serovars in five species of livestock in Thailand and to identify associations between seropositivity and sex, age, species and geographical locations. Sera from 14188 livestock (9288 cattle, 1376 buffaloes, 1898 pigs, 1110 sheep, 516 goats) from 36 provinces were tested for antibodies against 24 Leptospira serovars with the microscopic agglutination test (MAT) for which the criterion for a positive result was set at a titre of ≥1:50. A total of 1635 [11·5%, 95% confidence interval (CI) 11·0-12·0] animals were seropositive and the highest prevalence (30·4%, 95% CI 28·2-32·5) of evidence of infection was recorded in the northeast region followed by the central region (22·2%, 95% CI 20-24·6). Seroprevalences recorded for cattle, buffaloes, pigs, sheep and goats were 9·9% (95% CI 9·3-10·5), 30·5% (95% CI 28·1-32·9), 10·8% (95% CI 9·5-12·3), 4·7% (95% CI 3·6-6·1) and 7·9% (95% CI 5·8-10·5), respectively. Buffaloes were 3·1 (95% CI 2·8-3·4) times more likely than cattle to be seropositive. The most commonly detected antibodies were against L. interrogans serovars Ranarum, Sejroe, and Mini in cattle, Mini, Sejroe, and Bratislava in buffaloes, Ranarum, Pomona, and Bratislava in pigs and Mini, Shermani, and Ranarum in sheep and goats. Seroprevalences in cattle and buffaloes trended upwards with increasing age and there was no difference in the risk of seropositivity between males and females.