Applications of fluorescence in situ hybridization (FISH) for translocation studies and biological dosimetry would benefit substantially from reliable and efficient automatic detection of metaphase chromosomes labeled with fluorescent dyes. We replicated and evaluated a fluorescence metaphase finder previously developed at the Medical Research Council (MRC), Human Genetics Unit (Scotland) and at Lawrence Berkeley Laboratory (LBL; California). The MRC/LBL system seemed to detect nearly all of the metaphases on the test slides, but it presented an unacceptable number of false positives (about five false positives per one true positive). Furthermore, we determined that the system actually overcalled true detections by counting certain metaphase spreads twice (duplicates). Through modifications of the MRC/LBL system, we developed the Lawrence Livermore National Laboratory (LLNL) system, which minimizes the detection of duplicates, incorporates new detection features, uses a binary decision tree (BDT) for classification, and provides functionalities to improve scanning accuracy and improve the post-detection review. To test the new system, DAPI-stained preparations of metaphase chromosomes from blood lymphocytes of four unrelated donors were placed on slides in drops ranging from 7 mm to 20 mm in diameter. Drops contained between 5 and 200 scorable metaphases each. The LLNL system achieved approximately 90% detection of non-duplicated metaphases as verified by an expert cytogeneticist, with typically less than one false positive per every one true positive detected.