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Genotype by environment interaction (GEI) is a complex problem that complicates the barley selection and breeding process. The knowledge of the relationship between cereal phenology and climatic data is important for understanding GEI and the physiological pathways responsible for the interaction effect. The grain yield of twenty winter barley genotypes in six environments was observed. Factors influencing the variability were analyzed using a linear mixed model. The partial least squares regression (PLSR) model was applied to determine the most relevant environmental variables in certain stages of development that explained GEI effects. Biplot with environmental variables explained 43.7% of the GEI. The barley was generally the most sensitive to the environmental conditions (relative humidity, maximum temperature and its variation, sun hours, and precipitation) during the anthesis and filling stage (May) which caused GEI. Temperature variables did not show significance only in the vegetative phase. Different genotypes responded differently to environmental factors. Genotypes NS-525, NS-589, and J-103 were highlighted as widely adaptable, and Zajeˇcar was a suitable and reliable location for yield testing. The GEI information presented in this paper can be useful in traditional plant breeding and future breeding programs through molecular research of crop developmental genes and examination of physiological processes in two-row barley.