Passage of Iris yellow spot virus, Tospovirus sp., in its vector, as well as its spatial and temporal distribution in an onion (Allium cepa L.) field, have been described. However, little is known of the distribution of the virus within onion hosts. The temporal dynamics of Iris yellow spot virus titer in leaves of ‘Colorado 6’ and ‘Talon’ onion cultivars in a greenhouse were investigated with or without thrips by using double antibody sandwich enzyme-linked immuno-sorbent assay. There was no significant difference in titer between the two cultivars (P = 0.1224); however, titer was significantly greater with thrips than without (P < 0.0001). Plant leaves, divided into top, middle, and base sections, showed that virus titer significantly increased from 1.813 and 1.854 optical densities in top and middle, respectively, to 2.065 in base sections. Leaves of field-infected plants were categorized by age into older, intermediate, and younger leaves. In pre-bulb (6- to 8-leaf-stage) plants, Iris yellow spot virus was most frequently detected in intermediate leaves; however, there was no significant difference in virus titer between leaf age categories. In post-bulb (11 leaf-stage) plants, frequency of virus detection was almost the same in older and intermediate leaves (P = 0.7217) but significantly less in younger leaves. Virus titer was greater in younger, then intermediate, and older leaves, with optical densities of 2.778, 2.649, and 2.349, respectively. Virus was not detected in dead leaves, bulb scales, basal plates, or roots. Information on Iris yellow spot virus distribution in host tissues is vital in epidemiological studies, evaluations of germplasm, and quarantine control of this tospovirus.