Clinical hematology and biochemistry are recognized as useful aids in health diagnosis in birds. The Little Penguin (Eudyptula minor) is endemic to Australia and numbers are declining due to a number of factors including declining health. This 2-year study on both Bowen and Lion Island populations aimed at assessing the potential causes of their declining health. Blood was collected from a total of 294 adult Little Penguins. R.B.C., Hb, P.C.V., M.C.V., M.C.H., M.C.H.C., W.B.C. and differential counts, thrombocytes and T.P.P. were measured. Multiple regression was used to identify relationships between hematological values and the following predictor variables: site, season, sex, time held prior to sampling and body condition. Values obtained showed some differences compared to the values for other penguin species reported in the literature. P.C.V., R.B.C., Hb, M.C.H.C. and T.P.P. showed major differences in comparison with a previous study on little penguins. Considering the influence of predictor variables, it appeared that: P.C.V., R.B.C., M.C.V., M.C.H., M.C.H.C. varied according to seasons; P.C.V., R.B.C., M.C.H., T.P.P. were correlated to body condition; P.C.V. was higher in birds on Bowen Island compared to birds on Lion Island; and T.P.P. was higher in females. These ecological and biological predictor variables have a strong influence upon hematological values. As a consequence, they must be taken into consideration when interpreting results of future studies.