MOTIVATION: The application of genome-wide chromosome conformation capture (3C) methods to prokaryotes provided insights into the spatial organization of their genomes and identified patterns conserved across the tree of life, such as chromatin compartments and contact domains. Prokaryotic genomes vary in GC content and the density of restriction sites along the chromosome, suggesting that these properties should be considered when planning experiments and choosing appropriate software for data processing. Diverse algorithms are available for the analysis of eukaryotic chromatin contact maps, but their potential application to prokaryotic data has not yet been evaluated. RESULTS: Here, we present a comparative analysis of domain calling algorithms using available single-microbe experimental data. We evaluated the algorithms' intra-dataset reproducibility, concordance with other tools and sensitivity to coverage and resolution of contact maps. Using RNA-seq as an example, we showed how orthogonal biological data can be utilized to validate the reliability and significance of annotated domains. We also suggest that in silico simulations of contact maps can be used to choose optimal restriction enzymes and estimate theoretical map resolutions before the experiment. Our results provide guidelines for researchers investigating microbes and microbial communities using high-throughput 3C assays such as Hi-C and 3C-seq. AVAILABILITY AND IMPLEMENTATION: The code of the analysis is available at https://github.com/magnitov/prokaryotic_cids. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.