This study characterized the diversity of 367 barley collections from 27 different countries, including 5 control cultivars, using several phenotypic traits. Morphological traits, including spike type, grain morphology, cold damage, and lodging rate, exhibited wide variations. Eighteen accessions matured early, while four accessions had longer culm and spike lengths than the controls. The ranges of total phenolic content (TPC), β-glucan content, ABTS•+ scavenging activity, DPPH• scavenging activity, and reducing power (RP) were 1.79-6.79 mg GAE/g, 0.14-8.41 g/100 g, 3.07-13.54 mg AAE/100 g, 1.56-6.24 mg AAE/g, and 1.31-7.86 mg AAE/g, respectively. Betaone, one of the controls, had the highest β-glucan content. Two accessions had β-glucan levels close to Betaone. Furthermore, 20 accessions exhibited increased TPC compared to the controls, while 5 accessions displayed elevated ABTS•+ scavenging activity. Among these, one accession also exhibited higher DPPH• scavenging activity and RP simultaneously. Based on the statistical analysis of variance, all the quantitative traits were significantly affected by the difference in origin (p < 0.05). On the other hand, grain morphology significantly affected biochemical traits. Multivariate analysis classified barley accessions into eight groups, demonstrating variations in quantitative traits. There were noteworthy correlations between biochemical and agronomical traits. Overall, this study characterized several barley varieties of different origins, anticipating future genomic research. The barley accessions with superior performances could be valuable alternatives in breeding.