To identify potential infectious agents in cases of seasonal hyperacute panuveitis syndrome (SHAPU) from vitreous biopsies of patients with this disorder. A retrospective cohort analysis. Vitreous biopsies were obtained during the course of care from 53 subjects with SHAPU. DNA extraction and whole genome shotgun sequencing was performed using Oxford Nanopore long read sequencing. Sequences were matched against microbial and human databases. Visual outcomes at presentation and at 6 months were recorded. Identification and characterization of metagenomic sequences in vitreous isolates from subjects with SHAPU. Adequate DNA for sequencing was obtained from 32 SHAPU subjects. Fifteen samples yielded bacteria on culture, with 14 S. pneumoniae and 1 S. aureus isolate recovered. Bacterial DNA was detected by whole genome sequencing in 29 of 32 cases. S. pneumoniae was the predominant organism recovered. Bacterial genomic loads ranged up to 10 000 bacteria/human cell, indicating active infection. No pathogens were detected in control samples. Reconstruction of bacterial genome was possible in 7 SHAPU cases and indicated diverse S. pneumoniae subtypes associated with individual cases. Sufficient DNA remained for analysis of torque teno virus by qualitative polymerase chain reaction in 17 cases, of which 13 were positive. Visual outcomes were mixed, with 7 patients having hypotonous eyes at 6 months, but 8 patients having better than 20/200 vision. No relationship could be discerned between presenting bacterial load and visual outcome. The majority of SHAPU cases show molecular evidence for concurrent S. pneumoniae infection. Good visual results are possible in treating SHAPU as endophthalmitis. The authors have no proprietary or commercial interest in any materials discussed in this article.