Abstract: Barley (Hordeum valgue L.) is a feed crop with high quality and energy, and defoliation is a regular method for the management of pasture. To study the influence of defoliation on barley green forage usage, four representative barley varieties (lines) from diverse ecological regions were used in this study to analyze the effects of different cuts and defoliation timing on the yield and quality of barley forage. The results indicate both forage yield and quality in all varieties are higher in the first cut than in the second. Repeated defoliation results in forage yield loss in all varieties among which cultivar ‘Yanfeng 1’ presents tolerant with the smallest decline percentage of 13%. Repeated defoliation leads to the increase in the ratio of dry weight to fresh weight and crude ash content, and decrease in the content of crude protein and crude fat in all varieties. The content of crude fiber, Ca and P varied in different varieties following repeated defoliation. Barley grass removed in tillering stage has higher nutrition contents than in filling stage, and is palatable to be directly eaten by animals, and the defoliation timing is good for the regrowth. Thus, repeated defoliation can be initiated in the tillering stage and varieties with good tolerance should be selected. Barley plants can be removed as fresh green forage in tillering stage and silage in filling stage, respectively.