A genome scan, searching for quantitative trait loci (QTL) for the traits cold tolerance and body weight in tilapia, was performed on a cross between a (Oreochromis niloticus×Sarotherodon galilaeus) male and a (O. mossambicus×O. aureus) female. Fifty-four microsatellites and 23 amplified fragment length polymorphism (AFLP) primer combinations were genotyped and tested for marker–trait associations. Sex-specific linkage maps were constructed from this data. Twenty-three point-wise significant marker–trait associations were found in the genome scan, and putative QTL were subsequently tested in another (On×Sg) × (Om×Oa) family. None of the putative QTL from the first experiment were significant in the second experiment. However, one microsatellite, UNH130, found to be associated to weight in the first experiment, was found to be strongly associated to cold tolerance in the second experiment. Since QTL for cold tolerance and body weight were recently found on the linkage group containing UNH130 (linkage group 23) in another study, this linkage group was investigated more closely using interval mapping. The results provide indications, but not conclusive evidence, of a QTL for cold tolerance on linkage group 23.