Zebrafish genome sequencing project has improved efficiency of positional cloning in zebrafish and traditional chromosome walking by isolating large insert genomic libraries has become a past. However, the genetic principles underlying the positional cloning still form the foundation for current chromosome walking using the genome sequence assemblies and related genomic sequence and clone information. This guide intends to summarize our accumulated experience in positional cloning using the current genomic resources and tools, and provide a practical guide to positional and/or candidate cloning of mutants of interest. Copyright © 2011 Elsevier Inc. All rights reserved.