High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx, which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment, low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However, whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here, we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50 mW for 355 nm and 20 mW for 444 nm excitation. Furthermore, the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably, human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype, and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition, multicolor FISH (mFISH) with human chromosome painting probes to 13,18, 21, and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together, our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover, a FISH analysis confirmed high purity of the sorted chromosomes. Additionally, in contrast to centromeric satellite probes, chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations, such as deletions and translocations, in prenatal diagnosis. textcopyright 2016 International Society for Advancement of Cytometry.