A desirable tester that elicits greater genetic difference in Striga resistance among test crosses in a breeding program has not been reported. Therefore, this study was conducted to characterize 30 Striga resistant yellow endosperm maize inbred lines and three testers with varying resistance levels to Striga using DArTseq SNP markers and agronomic traits to identify a suitable tester for resistance hybrid breeding. Marker-based and agronomic trait-based genetic distances were estimated for yellow endosperm maize inbred lines and testers with varying resistance levels to Striga. The Marker-based cluster analysis separated the Striga resistant lines and testers into two distinct groups. Although the susceptible tester (T3) was the most distantly related to the 30 Striga resistant inbred lines, it exhibited a narrower range in genetic distance estimates and poor agronomic performance under Striga infestation in crosses with the resistant lines. In contrast, the resistant tester (T2) showed a broader range in genetic distance estimates in pairs with the 30 resistant lines. Also, it formed many high yielding hybrids with desirable traits under parasite pressure. Furthermore, the most significant positive association between agronomic trait-based and marker-based distance estimates (r = 0.389, P = 0.01) was observed when T2 has paired with the Striga resistant maize inbred lines. It thus appears that T2 may be used as a suitable tester to determine the breeding value of lines in hybrid maize resistance breeding programs. T2 was the most suitable tester, with a tolerant tester (T1) as an alternative tester to characterize the combining ability of Striga resistant maize inbred lines. This result can also encourage other breeders to investigate testers relative discriminating ability with varying levels of resistance in hybrid breeding for resistance to diseases, pests, and other parasitic plants.