Fluorescence in situ hybridization (FISH) provides an efficient and powerful technique for ordering loci both on metaphase chromosomes and in less condensed interphase chromatin. Two-color metaphase FISH can be used to order pairs of loci relative to the centromere; two- and three-color interphase FISH can be used to accurately order trios of loci spaced within 1 Mb relative to one another. Loci separated by a distance > 1-2 Mb exhibit chromatin loops that often give rise to a statistically significant but incorrect order. We derive Bayesian methods for selecting the best locus order based on microscopic evaluation for each of these types of FISH mapping data. We then describe how the results from several two- and three-locus analyses can be combined to evaluate the approximate posterior probability of a given multilocus order within the limits of the technology utilized. These methods directly address the question of interest: What is the probability that the inferred two-, three-, or multilocus order actually is correct? We illustrate our analysis methods by applying them to previously described FISH mapping data of 14 markers in the BRCA1 region on chromosome 17q12-q21. We also propose design strategies to order a group of closely spaced (< 1 Mb) loci, two and three loci at a time, using a bisection strategy for two-color FISH data and a trisection strategy for three-color FISH data. These strategies have the best worst-case performance for ordering a new locus relative to a group of ordered loci and are nearly optimal for ordering a group of loci of unknown order. These, in conjunction with physical mapping strategies, provide efficient and reliable methods for gene map construction by FISH.