Iris yellow spot virus (IYSV) causes a foliar disease in onion (Allium cepa L.) that results in a reduction in bulb size. Currently, no IYSV-tolerant or -resistant cultivar exists and a genetic source for tolerance or resistance has not been identified. Because other disease control methods are limited, host plant resistance offers the best hope to combat this disease. In this study, 13 winter-sown onion entries were screened for iris yellow spot (IYS) symptoms during the 2007 and 2008 cropping seasons. Twenty plants from each plot were observed and rated weekly during the growing seasons for straw-colored, necrotic lesions, typical of IYSV infection. Collected plant samples were assayed for IYSV by enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR). Visual rating was done using a scale of 1 to 9 with 1 representing no symptomatic tissue and 9 representing more than 50% tissue damage. Two different plant sampling methods were used in disease rating to determine their effect on mean severity and to correlate disease severity with absorbance values. Of the entries tested, plants of NMSU 05-33-1 exhibited a delay in symptom expression and lower IYSV levels relative to plants of other entries. Plants of ‘Denali’ and ‘Gelma’ appeared to be more susceptible to IYSV than plants of other entries. Plant selection within the plot over time did not influence disease rating values. When the same plants were rated and sampled for IYSV using ELISA, there was a strong, positive correlation between rating and absorbance values.