Sorghum is Ethiopia's main staple meal and the most significant nutritional security crop. Aflatoxin contamination in sorghum grains has been documented all across the world, including Ethiopia. Therefore, the use of resistant genotype is considered to be the most feasible means to mitigate the problem. Thus, 20 sorghum genotypes were evaluated in vitro for their reaction to aflatoxin contamination at Haramaya University School of Plant Sciences, crop protection laboratory. Mycotoxin analysis was done using enzyme-linked immunosorbent assay (ELISA). The findings showed that the sorghum genotypes tested for aflatoxin B1 responses had considerably varied responses. Long Muyera was identified as the most sensitive genotype to aflatoxin B1 contamination (34.1 g kg-1); with contamination levels significantly beyond the maximum tolerated limit (10 g kg-1). From the highland genotypes with a contamination level of 6.9 g kg-1, genotype W-5 (Weger-5) was relatively resistant to aflatoxin B1. Teshale genotype was the least resistant of the lowland sorghum genotypes examined. Except for genotype Long Muyera, highland sorghum genotypes were less sensitive to aflatoxin B1 contamination than lowland sorghum genotypes. As a result, it is worthwhile to choose and use genotypes that remain resistant in certain areas.