Despite the advances in canine medicine and the rapid gaining of attention of canine models in biomedical field and particularly in hemoglobin genes research, the studies on canine hemoglobin composition are sparse with ambiguous findings. Our aim was: i) to investigate the electrophoretic pattern of canine hemoglobin and the possible effects of age, sex, and anemia using a capillary electrophoresis assay, and ii) to validate this assay and calculate reference intervals (RIs) for canine hemoglobin fractions. Blood samples were collected from 53 healthy and 42 dogs with regenerative and non-regenerative anemias. The Sebia Capillarys 2 flex-piercing was used for hemoglobin analysis and it was validated using canine blood samples. R statistical language was employed for the statistical analyses. A major hemoglobin fraction (named HbA0) and a minor one (named HbA2) were identified in 100% and 47.4% of samples, respectively. The within-run and between-run CV was 0.1% for HbA0, and 9.1% and 11.2% for HbA2, respectively. The extremely narrow range of HbA0 and HbA2 values hampered a linearity study using canine blood samples. The RIs for HbA0 and HbA2 were 98.9-100% and 0-1.1%, respectively. HbA0 and HbA2 values were not significantly correlated with age (P = 0.866) or reticulocyte count (P = 0.731). No differences were observed in the median HbA0 and HbA2 between the two sexes (P = 0.887), and healthy and anemic dogs (P = 0.805). In conclusion, the capillary electrophoresis revealed a major hemoglobin fraction and an inconsistently present minor fraction. No effect of age, sex, anemia, or regenerative status of anemia was detected. The assay used was validated and RIs were generated, so as to be suitable for use in future investigations.