Trifolium subterraneum L. subsp. yanninicum is a pasture legume that is widely grown in medium and high rainfall areas of southern Australia and shows waterlogging tolerance. This study investigated diversity within subsp. yanninicum corresponding to eco-geographic variables, which may help to identify adapted parents with new traits for genetic improvement. Diversity for 10 morphological traits, flowering time and leaf isoflavone content was investigated using 108 ecotypes derived from wild Mediterranean populations and 10 cultivars, grown as spaced plants. Among the ecotypes, the range of flowering time was 94–149 days after sowing, and contents of formononetin, genistein and biochanin A were 0.05–1.38%, 0.73–2.33% and 0.15–2.10% of dry matter, respectively. Leaf markings also varied considerably. Leaf size and petiole length were correlated at each growth stage. Later flowering genotypes had larger leaves, longer petioles, longer internodes and thicker stems at flowering, but smaller leaves and shorter petioles at both 63 and 88 days after sowing. Contents of genistein and biochanin A were unrelated, but both were negatively associated with formononetin. Flowering time had a weak positive influence on genistein and biochanin A, but a weak negative influence on formononetin. All traits among the ecotypes (except stem diameter and leaf mark crescent size) were significantly correlated with at least one of 22 eco-geographic variables from their collection sites. Precipitation and altitude were more influential than temperature. The study found sufficient diversity to broaden the narrow genetic base of current subsp. yanninicum cultivars; however, other agronomically important traits also need to be considered. Further diversity may result from targeted collection, particularly in areas not represented in annual legume genebanks.