Mungbean yellow mosaic India virus (MYMIV) infecting soybean and other legumes causes yellow mosaic disease (YMD). Evaluation of soybean genotypes for YMD resistance involves field screening at disease hot spots or in a protected environment using infectious clones or viruliferous whiteflies as sources of virus inocula. Development of efficient virus inoculation and quantification protocols to screen soybean genetic stocks against YMD is imperative for breeding resistant varieties. Binary plasmids harbouring complete, tandem dimeric genomic components DNA A and DNA B of MYMIV-soybean isolate were engineered. The infectivity of the clones was demonstrated in soybean genotypes JS335 and UPSM534 that display contrasting YMD resistance. As a follow-up, soybean germplasm lines, breeding lines, and representative cultivars that were initially screened at an YMD hot-spot were then subjected to Agrobacterium-based infection with MYMIV. Quantitative real time polymerase chain reaction (qRT-PCR) based copy number analysis of MYMIV genomic components allowed soybean genotypes to be classified into three discrete categories; resistant, moderately resistant and susceptible to the viral infection. Thus, a soybean germplasm disease screening system based on agro-infection and qRT-PCR based quantification of MYMIV was developed to facilitate breeding YMD resistant soybean. The implications of this study for obtaining YMD resistant soybean cultivars are discussed.