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The purpose of this study was to perform a quality assessment and provide a scientific-based checklist for prevalence studies on root and root canal anatomy by appraising the methodological quality of in vivo studies using cone-beam computed tomographic (CBCT) imaging as the assessment tool.A systematic assessment of the literature was conducted, and 211 studies were selected and submitted to a methodological evaluation. Data were grouped into categories such as journal impact factor, open access availability, language, study origin, journal publisher, sample size, and CBCT settings. Interrater agreement was calculated by applying the Holsti method and Cohen kappa. The Kruskal-Wallis test and Pearson correlation coefficient were undertaken to assess the different variables (α = 0.05).The included studies (N = 211) reported data on 247,616 teeth from 41 countries. The maxillary first molar was the most studied tooth (n = 69) and the second mesiobuccal canal the most investigated anatomic feature. The highest scores were associated with high-impact journals (r = 0.482, P < .05), large sample sizes (r = 0.374, P < .05), non–open access availability (P < .05), and English-based language (P < .05), but geographic region and journal publisher also had an impact on quality scores. The identified methodological gaps were used to formulate a scientific-based checklist for this type of study.Although a small improvement in the global quality of the studies was observed over the years, only less than half of the studies correctly addressed the participant recruitment and frame and had an adequate sample size or provided sufficient CBCT imaging settings. The proposed checklist highlights the most pertinent points to guide researchers throughout the experimental design and the implementation of epidemiological cross-sectional studies of this nature.