For years, anti-Leishmania immunoglobulin G (IgG) antibodies have been detected in the sera of dogs living in areas of leishmaniasis endemicity. They have also been found in the aqueous humor and cerebrospinal fluid. In contrast, a review of the literature failed to identify the detection of anti-Leishmania antibodies in urine samples from dogs with leishmaniasis. Ninety-five dog urine samples were examined for the presence of anti-Leishmania antibodies by using a protein A enzyme-linked immunosorbent assay (ELISA). Twenty additional urine samples were collected from healthy dogs as controls. An IgG2 ELISA was performed on 26 urine samples found positive by the protein A ELISA. Twenty-three urine samples found positive to anti-Leishmania antibodies were tested for the local production of anti-Leishmania antibodies in the urinary tract by means of the urine antibody coefficient. Ten urine samples (and the corresponding serum samples) were compared by Western blot (WB) analysis. Thirty-five out of the 95 urine samples were found positive, 57 were found negative, and 3 were found inconclusive for antibody detection by the protein A ELISA. A high correlation between protein A and IgG2 levels was found in positive urine samples. Anti-Leishmania antibodies were present in the urine of dogs that had leishmaniasis, urinary protein/creatinine (U P/C) ratios of greater than one, and normal urinary sediment. A statistically significant correlation was observed between the U P/C ratios and the levels of anti-Leishmania antibodies in positive urine samples. In general, WB analysis and the urine antibody coefficient suggested that the presence of anti-Leishmania antibodies in urine was the consequence of an impairment of filtration of the glomerular barrier. However, in some dogs, WB analysis could be interpreted as suggesting that the presence of anti-Leishmania antibodies was caused, to a lesser extent, by local antibody production in the urinary tract. Antibody detection in urine could be a noninvasive method for leishmaniasis diagnosis and prognosis in dogs with glomerulonephropathies.