Human kallikrein-related peptidases are serine proteases, with diverse, yet poorly defined, roles in carcinogenesis. Recently, we have suggested that the dog may represent a suitable animal model for studying kallikrein (KLK) functions in cancer. The initial objective of the present study was to investigate KLK10 expression in cancerous and noncancerous canine mammary tissues. Our results revealed that the in silico predicted canine KLK10 mRNA sequence (GenBank accession No. XM_541467) is not accurate. Specifically, the predicted mRNA harbors a 183-bp segment, which constitutes part of (CANFA)KLK10 intron I, and a 756-bp sequence that corresponds to the nonpreviously characterized (CANFA)KLK9 coding region. In this report we experimentally determined the coding regions of canine KLK9 and KLK10, which were shown to exhibit high homology to their human counterparts and possess all KLK-defining features. For (CANFA)KLK9, other than the classical form of the gene, three more alternatively spliced variants bearing exon 3 skipping and/or intron I retention were identified. The classical form and variant 1 of KLK9, as well as KLK10, were found to be widely expressed among all tissues examined. Lower positivity rates were detected for KLK9 variants 2 and 3. None of these transcripts, however, showed any preferential or differential expression between tumor and normal tissues. Interestingly, (CANFA)KLK9 and (CANFA)KLK10 were concurrently expressed in almost all tissue samples; this suggests a possible participation of these serine proteases in a common proteolytic cascade. Overall, this is the first study that experimentally characterizes canine KLK9 and KLK10 and demonstrates their expression in normal and malignant tissues.