Background: We studied the diagnostic properties of the percentage of successive RR intervals differing by at least x ms (pRRx) as functions of the threshold value x in a range of 7 to 195 ms for the differentiation of atrial fibrillation (AF) from sinus rhythm (SR). Methods: RR intervals were measured in 60-s electrocardiogram (ECG) segments with either AF (32,141 segments) or SR (32,769 segments) from the publicly available Physionet Long-Term Atrial Fibrillation Database (LTAFDB). For validation, we have used ECGs from the Massachusetts Institute of Technology–Beth Israel Hospital (MIT–BIH) Atrial Fibrillation Database. The pRRx distributions in AF and SR in relation to x were studied by histograms, along with the mutual association by the nonparametric Spearman correlations for all pairs of pRRx, and separately for AF or SR. The optimal cutoff values for all pRRx were determined using the receiver operator curve characteristic. A nonparametric bootstrap with 5000 samples was used to calculate a 95% confidence interval for several classification metrics. Results: The distributions of pRRx for x in the 7–195 ms range are significantly different in AF than in SR. The sensitivity, specificity, accuracy, and diagnostic odds ratios differ for pRRx, with the highest values for x = 31 ms (pRR31) rather than x = 50 (pRR50), which is most commonly applied in studies on heart rate variability. For the optimal cutoff of pRR31 (68.79%), the sensitivity is 90.42%, specificity 95.37%, and the diagnostic odds ratio is 194.11. Validation with the ECGs from the MIT–BIH Atrial Fibrillation Database confirmed our findings. Conclusions: We demonstrate that the diagnostic properties of pRRx depend on x, and pRR31 outperforms pRR50, at least for ECGs of 60-s duration.