Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies， mostly solid tumors， due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues， CTAs are tantalizing immunotherapy targets， since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers， and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this， the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides， which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumor and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA， IGF2BP3， had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2， SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies， but also at high levels in critical tissues， including kidney， brain and marrow， diminishing their utility. A more tissue-restricted candidate， NT5C1B， was detected in T-cell lymphomas， but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach， proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumors and normal tissues. This article is protected by copyright. All rights reserved.