Atlantic Cod (Gadus morhua) has lost the major histocompatibility complex class II presentation pathway. We recently identified CD8-positive T cells, B cells, and plasma cells in cod, but further characterization of lymphocyte subsets are needed to elucidate immune adaptations triggered by the absence of CD4-positive T lymphocytes. Here we use single-cell RNA sequencing to examine the lymphocyte heterogeneity in Atlantic cod spleen. We describe five TCR-positive T cell subsets and eight BCR-positive B cell subsets and propose a B cell trajectory of differentiation. Notably, we identify a significant subpopulation of T cells that are CD8-negative. Most of the CD8-negative T lymphocytes highly express the homolog of monocyte chemotactic protein 1b, and another subset of CD8-negative T lymphocytes express the homolog of the scavenger receptor m130. Uncovering the multiple lymphocyte cell sub-clusters reveals the different immune states present within the B and T cell populations, building a foundation for further work.