Canine milk protects puppies against infectious diseases through a variety of mechanisms. In this study, the presence of potentially probiotic lactic acid bacteria grown on MRS-Cys agar plates from milk of nine bitches was investigated. The Gram positive catalase negative bacilli identified in this study belonged to four Lactobacillus species (Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus murinus, Lactobacillus animalis), as well as one isolate that was identified as Weissella viridescens. Random amplified polymorphic DNA profiling revealed 28 different genetic profiles among the lactobacilli isolates. Their probiotic potential was evaluated through different assays, including survival in conditions that resemble those existing in the gastrointestinal tract, production of antimicrobial compounds, adherence to intestinal mucin, degradation of mucin and pattern of antibiotic sensitivity. Some strains showed potential for future applications as canine probiotics.