Fluorescence in situ hybridization (FISH) to pachytene chromosomes is used to support the international effort to sequence the tomato (Solanum lycopersicum) genome. Most of the FISH is being performed in Wageningen and in our lab in Fort Collins. We use pachytene chromosomes rather than metaphase chromosomes for several reasons. Pachytene bivalents are 10-15X longer than metaphase chromosomes so BAC placement is more accurate. Eu- and heterochromatin can be distinguished on pachytene chromosomes. Most pachytene bivalents are morphologically distinguishable. And pachytene bivalents have four copies of target DNA juxtaposed rather than two. At the beginning of the sequencing project, the plan was to capture most tomato genes by sequencing the DNA in euchromatin that comprises only 25% of the genome. BACs from tomato libraries were localized by FISH to differentiate BACs in eu- vs. heterochromatin, to define eu- heterochromatin boundaries, and to identify BACs appropriate to use as anchors for contigs. Subsequently, new sequencing techniques have made it practical to sequence the whole tomato genome, including DNA in heterochromatin. As contigs and scaffolds have been lengthened, FISH has taken on additional roles. FISH is being used to orient and position contigs and scaffolds on chromosomes independent of the tomato linkage map. This is especially important in pericentric heterochromatin where crossing over is largely suppressed. In addition, FISH is being used to determine the size of unsequenced gaps between adjacent scaffolds. For these purposes, the US group so far has localized 381 BACs, including BACs on all 24 tomato chromosome arms.