Workplace burnout among healthcare professionals is a critical public health concern. Few studies have examined organizational and individual factors associated with burnout across healthcare professional groups. The purpose of this study was to examine the association between practice adaptive reserve (PAR) and individual behavioural response to change and burnout among healthcare professionals in primary care. This cross-sectional study used survey data from 154 primary care practices participating in the EvidenceNOW Heart of Virginia Healthcare initiative. We analysed data from 1279 healthcare professionals in Virginia. Our sample included physicians, advanced practice clinicians, clinical support staff and administrative staff. We used the PAR instrument to measure organizational capacity for change and the Change Diagnostic Index© (CDI) to measure individual behavioural response, which achieved a 76% response rate. Logistic regression analysis was used to estimate the effects of PAR and CDI on burnout. As organizational capacity for change increased, burnout in healthcare professionals decreased by 51% (OR: 0.49; 95% CI, 0.33, 0.73). As healthcare professionals showed improved response toward change, burnout decreased by 84% (OR: 0.16; 95% CI, 0.11, 0.23). Analysis by healthcare professional type revealed a significant association between high organizational capacity for change, positive response to change and low burnout among administrative staff (OR: 2.92; 95% CI, 1.37, 6.24). Increased hours of work per week was associated with higher odds of burnout (OR: 1.07; 95% CI, 1.05, 1.10) across healthcare professional groups. As transformation efforts in primary care continue, it is critical to understand the influence of these initiatives on healthcare professionals' well-being. Efforts to reduce burnout among healthcare professionals are needed at both a system and organizational level. Building organizational capacity for change, supporting providers and staff during major change and consideration of individual workload may reduce levels of burnout.