We report here the construction of a genetic linkage map and an overlapping set of clones containing DNA markers linked to the causative locus for facioscapulohumeral muscular dystrophy (FSHD) on 4q35. Multipoint linkage analysis placed eight loci in the following order with odds greater than 1000:1: cen-D4S171-FXI-D4S426-D4S187-D4S130-D4S 163-D4S139-D4F35S1-qter. The most likely position of D4S809 was distal to D4F35S1. Thirty-four yeast artificial chromosomes (YACs) were isolated by PCR-based assays for STSs derived from DNA markers with known genetic and physical order. Walking from the insert ends of 2 YACs identified 7 additional YACs, bridging the gaps between three of the markers. Two new YACs were found by hybridization of a cosmid inter-Alu PCR product to dot blots of inter-Alu PCR products of YAC DNA pools. All YAC clones were positioned using the genetic and physical order of the STSs and inter-Alu PCR fingerprint data. Eleven of the YACs and two cosmids were mapped by fluorescence in situ hybridization to confirm the location of the clones and to detect chimerism. The 43 YACs were assembled into two contigs. The larger contig spans approximately 2.4 Mb and contains markers closest to the FSHD gene.