Objective: To characterize baseline canine lymphocyte phenotypes including lymphocytes coexpressing multiple markers by novel 7-color multiparameter flow cytometry. Study Design: Fresh canine peripheral blood lymphocytes of 79 healthy 26-week-old Beagle or Beagle-mix dogs were stained and analyzed. Results: The high number of samples and acquired flow data (averaging 1.9x105 cells/sample) allowed the detection of minor lymphocyte subsets coexpressing multiple lymphocyte markers. The averaged percentages of major lymphocyte subsets of CD3+, CD4+, CD8+, CD21+ and gd TCR+ cells from this study were 74.0, 43.6, 14.3, 9.6, and 0.2, respectively, which were comparable but uniquely different from other reports as they were simultaneously detected in the same sample. We demonstrated that the commonly used CD21 and CD3 monoclonal antibody (mAb) clones, previously recommended not to be used in the same staining, could and should be used together with the proper steps of lymphocyte gating. We found a high percentage (10.3%) of unidentified CD21–CD3+CD4–CD8–gdTCR– lymphocyte subset that has never been reported. The intensive gating strategy and the mean percentages of each lymphocyte subset to their parent subsets and to the total lymphocyte population are presented and discussed. Conclusion: The canine lymphocyte phenotypes were fully characterized. This novel multiparameter flow cytometry method is a powerful approach to in-crease the accuracy of lymphocyte phenotyping in dogs.