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Objective Care guidelines for cystic fibrosis (CF) recommend annual screening for anxiety and depression using standardised measures, the Patient Health Questionnaire (PHQ-9) and the Generalised Anxiety Disorder Scale (GAD-7). Research in other chronic illness groups such as diabetes has demonstrated that illness-specific distress predicts daily functioning above and beyond depression alone. To address the need for a measure of illness-specific distress, we developed and validated the CF Stress Questionnaire (CFSQ), which could serve as a meaningful adjunct to mental health screening.Methods We developed a CF-specific measure of perceived stress with multiple phases of input from our patient population. We then conducted a multisite CFSQ validation study with 200 adults with CF across 3 geographically diverse CF centres, to examine the CFSQ’s factor structure, internal consistency, convergent validity and test–retest reliability.Results Results of item and subscale-level analyses indicate that all but one subscale met the established internal consistency cut-off of >0.6. In terms of convergent validity, the CFSQ and its subscales were moderately to highly correlated with the PHQ-9 (r=0.73 for total score, p<0.05) and the GAD-7 (r=0.66 for total score, p<0.05) and moderately correlated with quality of life as measured by the Cystic Fibrosis Questionnaire Revised (CFQ-R) Social (r=−0.59 for total score, p<0.05) and Treatment Burden subscales (r=−0.63 for total score, p<0.05). Subscales of the CFSQ were moderately correlated with the CFQ-R Emotional or Physical Functioning subscales, and weakly correlated with forced expiratory volume in 1 s per cent predicted or body mass index.Conclusions The CFSQ is a valid and reliable measure in terms of internal consistency and convergent validity with existing measures of mental health and quality of life commonly used in CF care and research.