Objective: The objective of the study was to develop the Barriers to Physical Activity and Sport Questionnaire for lesbian, gay, bisexual, transgender and queer/questioning (BPASQ-LGBTQþ [encompassing all spectrums of sexuality and gender]) persons (LGBT), which measures barriers using a socio-ecological model, and to validate it through exploratory and confirmatory factor analysis. Study design: Validation study. Methods: First, content validity was achieved by (a) developing a bank of items, (b) discussing the adequacy of the items in a committee of experts and classifying the selected ones under three socioecological levels (intrapersonal, interpersonal, environmental) and (c) refining wording and relevance of the items after a pilot test. Second, 709 LGBTQþ persons completed the questionnaire online to establish construct validity, criterion validity and internal consistency. Results: Exploratory factor analysis revealed four underlying factors in the BPASQ-LGBTQþ, thus suggesting that the items initially considered as 'environmental barriers' could be constituting two separate factors based on social interactions or organizational aspects. Competing three- and four-factor models were tested using confirmatory factor analysis. The four-factor structure with two different factors accounting for environmental barriers obtained better values in all fit indices. Cronbach's alpha ranged from 0.745 to 0.813. Participants engaging in regular physical activity and sports reported lower scores in all the barriers subscales than their counterparts. Conclusion: The BPASQ-LGBTQþ is valid and reliable to measure barriers to physical activity and sports in LGBTQþ people across the different socio-ecological levels. It could be especially useful for understanding the complex relationships between these barriers, which is of great relevance for the design and implementation of interventions addressed to encourage physically active lifestyles among LGBTQþ people.