Fluctuations in relative white cell proportions in milk from inflamed mammary glands can be determined using differential cell count. We hypothesize that infection of mammary tissue results in deviations of specific cell proportions, dependent upon the pathogen involved. Our objective was to evaluate the milk leukocyte differential (MLD) in mastitic and healthy quarters. Milk from 460 quarters in 115 Holstein cows was collected and lymphocytes, neutrophils, and macrophages were counted using QSCOUT milk analysis system (Advanced Animal Diagnostics, Morrisville, NC) which includes a fluorescence microscope. A sterile pooled milk sample was also collected from each cow for pathogen identification. Culture results were classified as no growth (NOG), gram-negative (GN), gram-positive (GP), or other (OTH), which included mycoplasma and prototheca. Quarters with total leukocyte count (TLC) ≥ 200 were classified as affected. The MLD data were arcsine transformed whereas TLC was reciprocal transformed and back transformed after analyses. Models included quarter health status and position and pathogen category nested within cow. Average (95% CI) TLC for healthy and affected quarters was 52,000 (47,460–56,170) cells/mL vs 830,000 (678,530–1,031,860) cells/mL (P < 0.0001). Proportions of neutrophils (53.4% vs. 61.9%), lymphocytes (17.0% vs. 14.8%) and macrophages (28% vs. 21.4%) were different for affected vs. healthy quarters (P < 0.0001). Regarding quarter position in healthy quarters, TLC was greater in RF quarters, followed by LR, RR, and LF (P = 0.05). MLD was associated with quarter position, with greater proportions of macrophages in the front quarters (P = 0.03). For affected quarters, TLC was greater when pathogen growth was detected; counts were greatest for OTH followed by GP, and GN (P = 0.01). MLD depended on pathogen: Neutrophil % was greatest in GP (67%) followed by NOG (59%), GN (58%) and OTH (57%; P = 0.009). Lymphocyte % was greatest in OTH (17%), followed by GP (15%), NOG (16%), and GN (7%; P = 0.00002). Macrophage % was greatest in GN (32%), followed by OTH (24%), NOG (23%) and GP (18%; P = 0.03). Differentiable patterns in the changes in relative leucocytes proportions may provide useful information for the identification of causal agent in mastitis cases.