BACKGROUND: The high-alcohol-drinking (HAD1/HAD2) and low-alcohol-drinking (LAD1/LAD2) rat lines, derived from the N/NIH rat, were developed by using a within-family selection and rotational breeding design for alcohol preference and alcohol consumption. Previously, a 20-cM genome screen identified quantitative trait loci (QTLs) on chromosomes 5, 10, 12, and 16 by using F2 progeny from HAD1 and LAD1 animals. METHODS: A total of 459 F2 HAD1 x LAD1 animals had been previously genotyped, and 428 HAD2 x LAD2 F2 animals were genotyped for microsatellite markers within the identified QTL regions. Linkage analyses were performed with the program QTL Express, a recently developed Web-based interface that implements a least-squares method. RESULTS: The linkage peaks previously identified in the HAD1 x LAD1 genome scan relied on one or two markers. Placement of additional markers in and around the QTL regions provided further support for each of the QTLs. Two of the QTLs on chromosomes 10 and 16 were confirmed in the replicate line; these QTLs exhibited linkage in both the HAD1/LAD1 and HAD2/LAD2 studies. CONCLUSIONS: This study demonstrated the importance of confirmation of QTLs in a replicate line, as well as the complexity of the genetic contribution to alcohol preference. Assessing these QTL regions in the inbred HAD/LAD animals will further facilitate characterization of these regions.