Polymorphisms of β-amylase among 19 species (27 taxa, 337 accessions) of wild barley, including Hordeum vulgare ssp. spontaneum (174 accessions), H. bulbosum (33), H. murinum (81), H. marinum (28), H. brachyantherum (4), H. jubatum (2), H. chilense (2) and H. roshevitzii (2) were studied using both isoelectric focusing (IEF) and thermostability analysis. Wide genetic variations were found. In general, the IEF patterns of H. vulgare ssp. spontaneum were markedly different from those of other wild species. Two new β-amylase IEF patterns (Ia and III) were observed in H. vulgare ssp. spontaneum in addition to the two patterns (I and II) found in cultivated barley (H. vulgare ssp. vulgare). In H. bulbosum, H. murinum, H. marinum and other wild species, 21 new IEF patterns were observed. Besides the A, B and C thermostability types reported in cultivated barley, new thermostability types (A+, A-B, B-C and C-) were frequently observed. Some accessions from H. arizonicum, H. jubatum, H. depressum and H. brachyantherum showed superior thermostability (A+). The genetic differentiation of β-amylase in relation to the phylogeny of genus Hordeum is also discussed.