Technical Abstract: Sunflower rust is a prevalent disease in many countries throughout the world and has been an increasing threat to the sunflower industry in North America in the last few years. Rust incidence increased from 17% in 2002 to over 70% of fields affected in the last two years. The USDA-ARS Sunflower Research Unit has released rust resistant breeding materials for several decades. However, constantly co-evolving rust populations have formed new virulent races to which current hybrids have little resistance. This study evaluated selected sunflower lines for resistance to race 336, the predominant race in North America, and to race 777, the most virulent race currently known. It also reconfirmed molecular markers linked to rust resistance genes. A total of 104 entries were tested for their reaction to races 336 and 777. Only 13 were resistant to both races, whereas another six were resistant only to race 336. The interspecific germplasm line, Rf ANN-1742, was resistant to both races and was identified as a new rust resistance source. A selection of 24 lines including 19 lines resistant to races 777 and /or 336 was screened with DNA markers linked to rust resistance genes R1, R2, R4u, and R5. The results indicated that the existing resistant lines are diverse in rust resistance genes. Durable genetic resistance through gene pyramiding will be effective for the control of rust.