The impact of leptospirosis on food animals and humans has not been fully elucidated. This study was designed to determine the prevalence of pathogenic organism in renal tissues of slaughtered cattle from two major abattoirs in southwestern Nigeria. One hundred and seventy (170) kidney samples of different breeds, ages, and sexes were collected and examined using cultural isolation, microscopic agglutination test (MAT), Warthin silver (WSs) impregnation and immunohistochemistry (IH). Chi-square test was used with a confidence level set at 0.05 to ascertain associations between positive cases and sex, age, and breed. One hundred and thirty (76.5%) of the kidney samples were culturally positive while 83.3% (25/31), 41.6% (37/89), and 35% (7/20) were MAT, WSss, and immunohistochemically positive, respectively. Interstitial nephritis (87%, 94/108) and tubular nephrosis (77.8%, 84/108) were the most prominent and consistent microscopic lesions observed.Serovars Hardjoprajitno (29%) and Bratislava (22.6%) were the most prevalent serovars. To the best of our knowledge, studies on determination of pathogenic serovars using cultural isolation, MAT, WSSs, and IH from kidneys of naturally infected cattle in Nigeria are rare. This study is the most comprehensive work in the detection of pathogenic Leptospira species from cattle in Nigeria.