Assessment of new genotypes in on-farm trials and under different tillage options is a current strategy for in-station experiments to enhance the breeding process and its final output in farmers’ fields, which can help increase productivity and sustainability in variable rainfed conditions. The objectives were to evaluate the agronomic performance of barley genotypes under different tillage systems in farmers’ fields and to provide suggestions to help farmers use resources more efficiently and sustainably for their proper field management. Five barley genotypes including four cultivars (Abider, Sararood1, Nader, Efes-3) and a promising breeding line (Yea168) were tested in three tillage methods, that is, conventional tillage (CT), reduced tillage (RT) and no tillage (NT) in on-farm trials in two locations (Sarabnilofar and Dalahoo) and three cropping seasons (2018–2021). The results revealed high variability in productivity and other studied traits in farmers’ conditions that were significantly affected by genotypes, tillage managements, locations, and years. Barley genotypes, except Efes-3, positively interacted with CT. The highest grain yield was observed in CT conditions (2613 kg/ha), followed by NT (2520 kg/ha) and RT (2470 kg/ha), showing about 5.8% and 3.6% better performance in CT than in RT and NT, respectively. Across locations and years, breeding line Yea168 outperformed all four cultivars in all three tillage systems and should be recommended for cultivation under rainfed conditions. The results of genotype by trait (GT) biplot analysis indicated that the traits relations and traits profiles of genotypes are different among tillage systems and locations. High-yielding genotypes had their own specific traits that resulted in their better performance. Breeding line Yea168, followed by Nader cultivar performed well in Sarabnilofar location, where 1000-kernel weight (TKW), plant height (PLH), number of grains per spike (NGPS), normalised difference vegetation index (NDVI) and spike per square metre (spike/m2) were the most important traits in deciding grain yield in this location, whereas in Dalahoo location, Sararood1 performed well and TKW, SPAD (chlorophyll content), NGPS, and spike/m2 had considerable contributions to grain yield. The findings demonstrated the breeding line Yea168 as the best-performing genotype across tillage systems, making it highly recommended for cultivation in rainfed areas of western Iran.