The aim of this study was to establish associations of kernel weight and colour with ecogeographic factors and molecular markers, based on ten wild barley [Hordeum spontaneum (C. Koch) Thell.] populations sampled in Israel across a southward transect of increasing aridity. Kernel weight and colour category were scored using barley kernels (naked caryopsis). Small kernel sizes (0.011 g kernel–1) and dark kernels were found in xeric populations. A higher variation of kernel weight was observed in xeric populations. A higher proportion of variation occurred within, rather than among, populations. Water, temperature and soil factors were associated with kernel size variation. Among 18 simple sequence repeats (SSRs) investigated, HVM14, HVM36, HVM43, BMS64 and BMS90 were associated with kernel weight, and HVM68 with kernel colour. The results indicated that high phenotypic variation and genetic diversity are related to ecological stress, and that the association of phenotypic traits with molecular markers, based on natural plant populations, should be interpreted cautiously due to the high chance of spurious associations between traits and molecular markers.