Densoviruses (DVs) are a group of viruses that contain a linear single-stranded DNA genome between 4–6 kb in length. Herein, we report a DV with a 5,480-nt genome, isolated from tobacco aphid (Myzus persicae nicotianae Blackman), named MpnDV. Unlike the genome of M. persicae densovirus (MpDV), which possesses five open reading frames (ORFs), the genome of MpnDV contains four putative ORFs—the nonstructural protein 1 (NS1) and NS2 from MpnDV are 98 - and 52-amino acids longer than those of MpDV, respectively, at the N-terminus, and the capsid proteins (VP) are 102 amino acids longer at the C-terminus than those of MpDV. Mapping of the MpnDV transcripts by RACE method indicated that the ORF of NS2 started at nt 340 and the right two putative ORFs were combined together by deleting two introns, one of 95 bp located at nt 2,932–3,026 and the other of 145 bp located at nt 4,715–4,859, suggesting transcript mapping was necessary for analyzing of genome organization. Alignment analysis indicated that MpnDV shows 97% sequence identity with MpDV, and that the shortened ORFs resulted from nucleotide indels, suggesting MpnDV and MpDV were two isolates of the same virus. Thus, MpnDV and MpDV clustered together in a tree-based analysis. The prevalence of MpnDV infection in wild populations of tobacco aphids differed among 29 locations; 34% of the 622 individuals sampled were positive. The genome organization, transcript strategy, and widespread distribution in wild populations suggest that MpnDV might possess a biological function different from that of MpDV.