Weight per thousand seeds (WTS) is an important quality character of barley grain. In recent years, some researchers have shown that WTS is affected by genetic factors, as well as by its cultivation and environmental conditions. However, the effects of different environmental factors on barley WTS remain unclear. In addition, although previous studies have mostly been conducted at several sites, large-scale studies have not systematically addressed the relationship between cultivated barley WTS and environmental factors. Moreover, with a large number of hull-less barley varieties or resources being required on an urgent basis for modern agriculture development in an environment of high radiation, low temperature, and drought, Qinghai-Tibet Plateau is an important region for growing hull-less barley in China. However, until now, the relationship between barley WTS and environmental factors in Qinghai-Tibet Plateau has not been reported. Therefore, the present study investigated the distribution of cultivated barley WTS characteristics in Qinghai-Tibet Plateau using geography, climate, soil, and agronomy data from 83 sampling sites. The aim of this study was to identify the relative effects of different environmental factors on WTS, to clarify the relationship between the distribution of barley WTS and environmental factors, and to explicitly assess acclimation of WTS of barley varieties planted in different areas to the environment. The results showed that (1) WTS exhibited a macrocosm pattern of spatial distribution along the horizontal direction in Qinghai-Tibet Plateau, with staggered patches of different values and an N-shaped tendency. In addition, two regions of higher WTS (south-western and north-eastern) included the counties of Qushui, Duilongdeiqin, Naidong, Shigatse, Zhanang, Gongga, Jiacha, Dazi, Qiongjie, Lazi, and Dingri in Tibet and Haiyan, and Mengyuan and Gangcha in Qinghai Province. (2) Along the vertical direction, GSC distribution exhibited a pattern with double peaks, with two higher value zones between the altitudes from 3600.0 m to 3900.0 m and that above 4500.0 m with average WTS values of (49.6815±10.0764) g and (47.9500±0.1732) g, respectively. (3) The effect of the magnitude of environmental factors on WTS followed an order of precipitation during the growth period from heading to maturation > soil available K content > sunshine hours during the growth period from tillering to elongation > average temperature during the growth period from heading to maturation > sunshine hours during the growth period from heading to maturation > average diurnal temperature range during the growth period from elongation to heading > longitude.