Background There is a great deal of variation in the existing capacity of primary prevention programs and policies addressing chronic disease to deliver evidence-based interventions (EBIs). In order to develop and evaluate implementation strategies that are tailored to the appropriate level of capacity, there is a need for an easy-to-administer tool to stage organizational readiness for EBIs. Methods Based on theoretical frameworks, including Rogers’ Diffusion of Innovations, we developed a survey instrument to measure four domains representing stages of readiness for EBI: awareness, adoption, implementation, and maintenance. A separate scale representing organizational climate as a potential mediator of readiness for EBIs was also included in the survey. Twenty-three questions comprised the four domains, with four to nine items each, using a seven-point response scale. Representatives from obesity, asthma, diabetes, and tobacco prevention programs serving diverse populations in the United States were surveyed (N = 243); test-retest reliability was assessed with 92 respondents. Results Confirmatory factor analysis (CFA) was used to test and refine readiness scales. Test-retest reliability of the readiness scales, as measured by intraclass correlation, ranged from 0.47–0.71. CFA found good fit for the five-item adoption and implementation scales and resulted in revisions of the awareness and maintenance scales. The awareness scale was split into two two-item scales, representing community and agency awareness. The maintenance scale was split into five- and four-item scales, representing infrastructural maintenance and evaluation maintenance, respectively. Internal reliability of scales (Cronbach’s α) ranged from 0.66–0.78. The model for the final revised scales approached good fit, with most factor loadings >0.6 and all >0.4. Conclusions The lack of adequate measurement tools hinders progress in dissemination and implementation research. These preliminary results help fill this gap by describing the reliability and measurement properties of a theory-based tool; the short, user-friendly instrument may be useful to researchers and practitioners seeking to assess organizational readiness for EBIs across a variety of chronic disease prevention programs and settings.