Thirty-four populations of Trifolium medium collected in central, northern and southern Poland were used in a morphological and enzymatic study. Twenty-two characters of leaf, stem and inflorescence were scored. The enzymatic survey included aspartate aminotransferase (AAT), esterase (EST), glucoso 6-phosphate isomerase (PGI), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (PGD), peroxidase (PRX), and shikimate dehydrogenase (SKD) assayed in two buffer systems. The populations showed a high intra- and inter populational variability both on morphological and enzymatic levels. The data demonstrate that morphological and enzymatic variability is regionally dependent in the same manner. Morphological traits, such as pedicel length, peduncle length, terminal leaf length and width and stipule length were correlated with the majority of climatic variables. PGI, PRX and LAP phenotypes were correlated with a set of climatic variables of high altitude areas. AAT, EST and PGD phenotypes were good predictors of more balanced climatic conditions. The altitude factor had a significant effect on all phenotypes but AAT. This study also showed that the same climatic variables did not always play a significant role in the morphological and enzymatic differentiation of T. medium. In general, enzymes were much more responsive to the variety of climatic factors than morphological characters.