BACKGROUND: The microscopic agglutination test (MAT), the standard method for serological diagnosis of leptospirosis, may present limitations regarding its sensitivity. Current studies suggest that Leptospira immunoglobulin-like (Lig) proteins and LipL32 are of particular interest as serodiagnostic markers since they are present only in pathogenic species of the Leptospira genus. The purpose of this study was to identify leptospiral immunodominant proteins that are recognized by canine sera from diseased dogs. RESULTS: A total of 109 dogs were studied, including seroreactive dogs (MAT ≥800) and dogs with no seroreactivity detectable by MAT. Eight recombinant fragments (31-70 kDa) of pathogenic Leptospira were tested for their use as diagnostic markers for canine leptospirosis using the Multi-antigen Print Immunoassay (MAPIA) platform: LigB [582-947aa] from L. interrogans serovar Pomona, L. interrogans serovar Copenhageni and L. kirschneri serovar Gryppotyphosa, LigB [131-649aa] from L. interrogans serovar Copenhageni, L. interrogans serovar Canicola and L. kirschneri serovar Gryppotyphosa, LigA [625-1224aa] L. interrogans serovar Copenhageni and LipL32 from L. interrogans serovar Copenhageni. The data were analyzed and ROC curves were generated. Altogether, LigB [131-649aa] L. interrogans Canicola, LigB [131-649aa] L. kirschneri Gryppotyphosa and LipL32 L. interrogans Copenhageni showed best accuracy (AUC = 0.826 to 0.869), with 70% specificity and sensitivity ranging from 89% to 95%. CONCLUSIONS: These results reinforce their potential as diagnostic candidates for the development of new methods for the serological diagnosis of canine leptospirosis.