ABSTRACT The beet necrotic yellow vein virus (BNYVV) is a multipartite virus with the highest number (up to five) of genomic segments among RNA viruses. Classified as a soil-borne virus, it is persistently transmitted by the protozoan Polymyxa betae . Previous studies have demonstrated that the relative frequency of the BNYVV genomic RNAs was modified depending on the host plant as well as the infected organ, resulting in distinct stoichiometric ratios between the viral RNAs. In this study, we investigate whether infection by the vector P. betae influences the relative abundance of BNYVV RNAs within the roots of the host plant Beta vulgaris . Furthermore, we examine the relative frequency of BNYVV genomic segments and the viral load of BNYVV at two different stages of P. betae ’s biological cycle: zoospore and resting spore. Our finding offers new insights into understanding the biology of this soil-borne virus and its vector. Notably, the variations in the relative accumulation of BNYVV RNAs observed in zoospores and resting spores, along with a higher viral load in zoospores compared to resting spores, invite consideration of the virus’s replicative capacity within the vector. IMPORTANCE Our understanding of the transmission of plant viruses by protozoan vectors remains poor and fragmented. The fate of viral elements in the living stages of the vector is unknown. Here, we first established a protocol allowing the purification of two forms of the vector free of cellular contaminants. This permitted the examination of the relative frequencies of beet necrotic yellow vein virus RNAs in the roots of its natural host and in two forms of its protozoan vector, Polymyxa betae , responsible for virus transmission. Our findings provide new insights into virus behavior during vector transmission, allowing us to analyze how the virus regulates its RNA frequencies and load within the vector. By focusing on the early stages of viral transmission and separating virus acquisition from transmission to new hosts, we pave the way for experiments aimed at elucidating the molecular mechanisms behind viral acquisition and the maintenance of viral genome integrity by P. betae .