Barley is traditionally grown in the Mediterranean region as a dryland crop, invariably under drought-stressed conditions and often without inputs such as fertilizer. Following research that demonstrated the benefits of fertilization, even under less-than-favourable rainfall condition, fertilizer use on cereals has increased dramatically in the past few decades in countries of West Asia-North Africa. With developments in breeding new barley cultivars for higher yield, combined with disease resistance and environmental adaptability, it is crucial to assess the extent to which such cultivars respond to fertilizer inputs as this may affect aspects of a breeding strategy, particularly the choice of germplasm for adaptation in any particular environment. Thus, we assessed the yield potential of 30 barley cultivars with a range of germplasm types, including new cultivars and landraces, in a greenhouse in two soil types with and without adequate nitrogen and phosphorus fertilizer, i.e., low and high fertility. By comparison with the unfertilized low fertility soils, the fertilizer treatment increased yield parameters by about 10-fold. However, the rankings of some cultivars changed markedly with fertilization; some increased, others showed poor responses and decreased relatively, and two performed well with and without fertilizer. Based on the differential responses at the initial screening stage in the greenhouse, it is possible to identify lines or cultivars that are highly responsive to fertilizers and to incorporate such germplasm for further development to produce high-yielding cultivars for commercial adoption by farmers.