Dogs are an important model species for the development of novel cellular immunotherapies, including adoptive NK cell therapies. Characterization of canine natural killer (cNK) cells has been hindered by a lack of species-specific reagents and an inability to efficiently isolate and expand these cells. Additionally, prior attempts to identify cNK cells have relied heavily on knowledge which may only be applicable to human NK cells. Our group is able to produce large numbers of innately cytotoxic canine lymphocytes. Here, we aim to provide novel characterization of these cells. Peripheral blood mononuclear cells were isolated from canine buffy coats and CD5+ T cells were depleted using magnetic separation. Depleted cells were cocultured with K562 cells expressing 4-1BBL and membrane-bound IL21 and supplemented with rcIL-2 and TGF-β for 21 days. On day 21, immunophenotyping and calcein cytotoxicity assays were performed. Based on these data, day 21 expanded cells were FACS sorted into four comprehensive immunophenotypes and expanded for an additional 7 days. On day 28, immunophenotyping and cytotoxicity assays were repeated. Over the course of expansion, CD8α and NKp46 expression increased and CD3, CD4, CD8β, CD21, and TCR expression decreased. Cytotoxicity was negatively correlated with CD3 and TCR expression and positively correlated with CD8α expression. There was no correlation between CD5, CD16A, or CD94 expression and cytotoxicity. FACS sorting confirmed that TCR-negative cells exhibit significantly more innate cytotoxicity against canine cancer cell lines when compared to αβ and γδ T cells. Additionally, expanded cells express higher levels of CD8α and lower levels of CD8β when compared to peripheral blood leukocytes. Our results suggest that cNK cells are characterized by high CD8α expression, likely in the form of CD8αα homodimers, and high NKp46 expression in the absence of putative T and B cell markers such as CD3 and CD5.