Barley crop production in Mexico in recent years has been affected by rust, of which yellow rust (YR) has regained its importance. This investigation aimed to identify the variability of YR and to evaluate the resistance level of seedlings and adult plants, the agronomic behavior of 15 advanced lines, and one control variety of two-row barley. During the 2021 spring-summer season, YR samples were collected from locations in the Highland Valleys of Central Mexico. Establishment of a field test in three locations allowed estimation of the agronomic behavior and resistance in adult plants; while evaluation of seedlings resistance was in a greenhouse. Nine YR isolations were identified, of which the most frequent (27%) was CMEX/21.9. Twelve lines expressed resistance in seedlings and adult plants. Alexia “s” is the candidate line with immunity to yellow rust. The results of resistance (field and greenhouse) and agronomic behavior allowed germplasm identification with outstanding characteristics. The release of varieties with genetic resistance to diseases contributes to the reduction of environmental deterioration.