This paper is the first study of the prevalence of leptospirosis in the cattle at slaughter from a rural area of Kazakhstan. Five hundred and seventy three samples of serum, urine, and kidneys from cattle of Alatau, Kazakh white and Auliyekol breed, aged from 2 to 5 years (unknown vaccination status), from the province of Almaty in the South-Eastern region were collected during four years (March 2010 to October 2013). The serological, bacteriological, and molecular analyses were performed. Serum samples were tested with 14 reference Leptospira serovars by microscopic agglutination test (MAT). MAT results showed that 89 (15.53%) serum samples had detectable antibodies against seven serovars of L. interrogans at a dilution of ≥1:100. Serovars: Pomona (38.2%), Tarassovi (27.2%), and Kabula (18.8%) were the most prevalent and their titres ranged from 100 to 1200. The spirochetes were detected in 11 samples of urine and nine samples of kidneys under dark-field microscope observation. The pure cultures were obtained from three samples. PCR technique confirmed leptospirosis in 23 out of 89 urine samples from cows, which showed the presence of leptospiral antibodies in microagglutination test. The high disease prevalence in cows indicates the high Leptospira contamination in this area. It was concluded that the bovine leptospirosis is an endemic and locally widespread disease in Kazakhstan, and that it may play a role in zoonotic transmission to humans.