Even though the Gallus gallus chromosome 16 (GGA 16) contains the major histocompatibility complex and is especially important in disease resistance in poultry, the gene map for GGA 16 is incomplete. Trisomy mapping using Cornell trisomy line chickens naturally exhibiting 2, 3 and 4 copies of GGA 16 is a powerful means for assigning genes to GGA 16. MHC-B, MHC-Y and CD1 genes were all previously assigned to GGA 16 by trisomy mapping. Here we combine trisomy mapping with array Comparative Genomic Hybridization (aCGH) to identify additional GGA 16 sequences among unassigned genomic sequence contigs. A custom 4x44K Agilent aCGH was developed that includes 40,000 probes with a spacing of about one 60mer probe for each 4Kb of unassigned genomic sequence. Also included were 500 GGA 16-specific positive control probes and 1,546 negative control probes specific for other autosomes and sex chromosomes. The DNA used in array hybridization included eight samples each from individuals within the Cornell trisomy line previously typed as disomic, trisomic, and tetrasomic for GGA 16. After scanning and feature extraction, data was normalized by scaling and centering methods. Significance was tested using mixed linear models comparing disomic individuals to tri- and tetrasomic individuals. The false discovery rate (FDR) was calculated. A total of 489 probes were found to be significant at a FDR of 10%, including 91 probes from unassigned contigs and 398 probes specific for GGA 16. These findings confirm our hypothesis that trisomy mapping and aCGH can be combined for assigning additional sequences to GGA 16 and have revealed a number of candidate genes for further study.