Peripheral blood leukocytes (PBL) are critical for understanding systemic immune responses and assessing the organism's health. In immunological studies, particularly with aquaculture-relevant species like Rainbow trout (Oncorhynchus mykiss), ensuring PBL viability during sample storage is important, especially when samples must be transported over long distances. Anticoagulants are essential for preventing blood clotting and preserving cellular integrity; however, their effects on leukocyte populations in salmonids remain poorly studied. This study aimed to evaluate the impact of four anticoagulants-ethylenediaminetetraacetic acid (EDTA), sodium citrate, sodium heparin, and lithium heparin-on the viability of PBL populations in rainbow trout, with a comparative analysis of human PBL. Blood samples were stored for up to seven days, and cell viability and leukocyte population percentages were assessed by flow cytometry at 0, 1-, 2-, 4-, and 7-days post-collection (dpc). Results showed that sodium heparin and lithium heparin were the most effective anticoagulants for preserving trout leukocyte viability, maintaining percentages greater than 70 % up to 4 dpc. In contrast, EDTA and sodium citrate were less effective in maintaining cell viability. In human PBL samples, EDTA was the most effective anticoagulant, with lymphocyte viability exceeding 80 % at 7 dpc. Sodium heparin and lithium heparin also preserved human PBL viability comparably up to 4 dpc, but their efficacy decreased significantly by 7 dpc. These findings highlight the species-specific effects of anticoagulants, recommending heparin-based anticoagulants for longterm leukocyte preservation in rainbow trout (up to 4 dpc). In addition, this study provides valuable information for blood handling protocols in immunological research.