Abstract Wheat blast disease is responsible for severe production losses. The disease resistance associated with the 2NS/2AS translocation is effective, but its level can be variable. In this study, we evaluated the presence of 2NS/2AS translocation in 310 advanced breeding lines from six crosses (Caninde 2/Milan, Milan/Caninde 2, Maringa/Milan, Milan/Maringa, Ciano79/Milan and Milan/Ciano79), and also studied their wheat blast reaction in the field to three virulent pathogen strains (P13-009, P14-031 and P14-039) collected in Paraguay. Advanced lines of two crosses (Caninde 2/Milan and Milan/Caninde 2) yielded the highest number of blast-resistant entries without 2NS/2AS translocation. Earlier studies have shown Caninde 2 to be a moderately susceptible line, which in combination with Milan, is probably adding non-2NS/2AS type resistance to these crosses. Our result indicates that such a resistance is based on several additive factors derived from multiple sources, which need to be explored further and also used to develop more durable wheat blast-resistant germplasm in the future.