Using next-generation sequencing (NGS) for full genomic characterization studies of the newly emerging avian orthoreovirus (ARV) field strains isolated in Pennsylvania poultry, we identified two co-infection ARV variant strains from one ARV isolate obtained from ARV-affected young layer chickens. The de novo assembly of the ARV reads generated 19 contigs of two different ARV variant strains according to 10 genome segments of each ARV strain. The two variants had the same M2 segment. The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains. Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains. These findings have demonstrated the first naturally occurring co-infection of two ARV variants in commercial young layer chickens, providing scientific evidence that multiple ARV strains can be simultaneously present in one host species of chickens.