Background People with multiple long-term conditions are more likely to have poorer health-related quality of life (HRQOL). Yoga has the potential to improve HRQOL. Gentle Years Yoga© (GYY) is a chair-based yoga programme for older adults. We investigated the effectiveness and cost-effectiveness of the GYY programme in older adults with multiple long-term conditions. Methods In this pragmatic, multi-site, open, randomised controlled trial, we recruited older adults aged ≥65 years with ≥2 long-term conditions from 15 primary care practices in England and Wales. Participants were randomly assigned to usual care control or a 12-week, group-based, GYY programme delivered face-to-face or online by qualified yoga teachers. The primary outcome was HRQOL (assessed with EQ-5D-5L) over 12 months. Secondary outcomes included anxiety, depression, falls, loneliness, healthcare resource use, and adverse events. Results Between October 2019 and October 2021, 454 participants were randomly assigned to the intervention (n=240) and control (n=214) groups. Seven GYY courses were delivered face-to-face and 12 courses were delivered online. The mean number of classes attended among all intervention participants was nine (SD 4, median 10). In our intention-to-treat analysis (n=422), there was no statistically significant difference between trial groups in the primary outcome of HRQOL (adjusted difference in mean EQ-5D-5L = 0.020 [favouring intervention]; 95% CI -0.006 to 0.045, p=0.14). There were also no statistically significant differences in key secondary outcomes. No serious, related adverse events were reported. The incremental cost-effectiveness ratio was £4,546 per quality-adjusted life-year (QALY) and the intervention had a 79% probability of being cost-effective at a willingness-to-pay threshold of £20,000 per QALY. Conclusions The offer of a 12-week chair-based yoga programme in addition to usual care did not improve HRQOL in older adults with multiple long-term conditions. However, the intervention was safe, acceptable, and probably cost-effective.