The enterotoxigenic Escherichia coli (ETEC) F4ac is a major cause of diarrhoea in newborn and young pigs. The locus for the intestinal ETEC F4ac receptor (F4acR) has been mapped to pig chromosome (SSC) 13q41 with known homology to human chromosome (HSA) 3q21 and q29. However, the causative gene and mutation(s) remain unknown. The aim of this study was to characterize gene-derived markers on SSC13q41 for fine mapping of the F4acR locus, and construct a high-resolution pig–human comparative map to select positional candidate genes for F4acR. Pig-specific sequence-tagged site markers were developed for 20 genes that are located in a 6.8-Mb region on HSA3q21 and q29, and a total of 34 single-nucleotide polymorphisms (SNPs) were identified in 14 of 20 markers developed. Eighteen markers were mapped to SSC13q41, while the other two markers (PLXNA1 and KLF15) were assigned to SSC13q32 and SSC7q13, respectively, by radiation hybrid mapping. This result showed that there was a small conserved segment on SSC7 corresponding to HSA3q21. A framework map comprising 18 markers on SSC13q41 was established, refining the synteny breakpoint on SSC13q41 to a region of 12.3 centiRay. The comparative radiation hybrid (RH) map revealed three interesting candidate genes for F4acR from the human genome, viz. MUC4, MUC13 and MUC20. Linkage analysis with six marker polymorphisms revealed that MUC4 had the most significant linkage with the F4acR locus.