Potato (Solanum tuberosum L.) is a co-staple food and a source of cash income for the smallholder farmers' in eastern Ethiopia; however, the productivity of the crop is constrained by biotic and abiotic factors. Of which viruses are considered to be major yield limiting factor, but information is lacking on the prevalence and distribution of potato viruses in major growing districts of eastern Ethiopia. This research is therefore initiated to assess and determine the status of six major and multiple potato viruses' infection at farmers' fields during the main rainy and irrigation seasons in four major potato-growing districts of eastern Ethiopia. The dot-blot assays performed for 1000 leaf samples collected from 20 farmers' fields during the two seasons indicated an overall high incidence (71.9% samples detected positive for at least one virus) of potato virus in the surveyed districts of the regions. The most prevalent (19.4%) type of infection was the mixture of all six potato viruses. Among the major viruses, the results also confirmed PVM as the most prevalent (57.3%) virus followed by PVY (48.1%), whereas PVA (45.1%), which was not commonly detected in the other regions of the country, was detected as third most prevalent virus in the current study. The comparison of the results of virus detection in the samples from two seasons indicated remarkable differences in the incidence and prevalence of viruses, where an increasing trend of virus incidence and mixed virus infections from 59 to 84.8% and 41.2 to 72%, respectively, were recorded from rainy to irrigation season. The two viruses; PVX and PVS, which were identified as least prevalent during rainy season, were ranked the 3rd and 4th most prevalent viruses after PVM and PVY during irrigation season. The detected high incidence of virus in potato plants during this first systemic research conducted suggested the importance of continuing the intensive survey and detecting the status of viruses' infection in potato growing districts of eastern Ethiopia. It is also suggested to create awareness among smallholder farmers about the management practices to reduce the effects of viruses’ infection on the productivity of potato in eastern Ethiopia.