Background Synovial hypoxia is a hallmark of rheumatoid arthritis (RA). Photoacoustic (PA) imaging, based on the use of laser-generated US, can detect the oxygenation status of tissue in individuals with RA. However, large studies are lacking, with few investigating the correlation between oxygenation status and disease activity. Purpose To measure synovial oxygenation status in participants with RA by using a multimodal PA US imaging system and to determine the correlation between PA imaging-measured oxygen saturation (SO2) and disease activity. Materials and Methods In this prospective observational cohort study, multimodal PA US imaging examinations were performed on small joints of consecutive participants with RA, who were treated at two outpatient rheumatology clinics from 2019 to 2021, and healthy controls. The SO2 values of the synovium were measured with dual-wavelength PA imaging and classified into three categories-hyperoxia, intermediate oxygenation status, or hypoxia-based on the signal coloration and clustering analysis of the SO2 values. The correlations of oxygenation status with power Doppler US (PDUS) scoring and clinical disease activity index were evaluated with one-way analysis of variance and the Kruskal-Wallis test with Bonferroni correction. Results A total of 118 participants with RA (median age, 55 years [IQR, 41-62 years]; 92 women) and 15 healthy control participants (median age, 37 years [IQR, 33-41 years]; 11 women) were included. The wrist synovium was categorized as hyperoxic in 36 participants with RA, of intermediate oxygenation status in 48 participants, and hypoxic in 34 participants. All control participants had hyperoxic synovial tissues. For participants with RA, hyperoxic synovium had more affluent Doppler US-depicted vasculature than those with hypoxia and intermediate oxygenation status (mean PDUS grade: hyperoxia, 2.7 ± 0.6 [SD]; intermediate, 1.3 ± 0.7; hypoxia, 1.1 ± 0.8; P < .001). Participants with intermediate status synovium had a lower clinical disease activity index than those with hypoxia (intermediate, 11.0 [IQR, 5.0-21.5] vs hypoxia, 26.0 [IQR, 18.0-39.0]; P = .001). Conclusion Photoacoustic imaging-detected hypoxia in thickened synovium correlated with less vascularization and higher disease activity in participants with rheumatoid arthritis. Clinical trial registration no. NCT04297475 © RSNA, 2022 Online supplemental material is available for this article.