Tumors vary widely in chromosomal level genome instability. To gain a better understanding of the underlying defects which foster specific types of aberrations, we investigated the response of cells of related genetic backgrounds to challenge with methotrexate. We studied mismatch repair deficient HCT116 cells, two derivatives also deficient in XRCC5 (HCT116 Ku86+/-) or BLM (HCT116 BLM-/-), and mismatch repair competent HCT116+chr3 cells. We show that colony formation occurred at a significantly higher frequency in HCT116 cells and HCT116 Ku86+/- cells compared to HCT116 BLM-/- and HCT116+chr3 cells. Visible colonies arose most rapidly in HCT116 Ku86+/- cells, whereas they formed most slowly in HCT116+chr3 cells. Copy number changes acquired by the methotrexate resistant HCT116 and HCT116 BLM-/- cells most often included whole chromosome gains or losses or no acquired copy number changes, whereas resistance in HCT116+chr3 and HCT116 Ku86+/- cells was associated with amplification of DHFR and copy number transitions leading to increased copy number of DHFR, respectively. The additional copies of DHFR were present on unstable chromosomes and organized as inverted repeats in HCT116+chr3 cells, while they were most often present as direct repeats in HCT116 Ku86+/- cells. These observations suggest that different mutational mechanisms promote drug resistance in these genetic backgrounds; mismatch repair deficiency in HCT116, high rates of chromosomal instability in HCT116 Ku86+/-, and low rates of chromosomal instability in HCT116+chr3. On the other hand, it appears that loss of BLM function suppresses the mismatch repair mutator mechanism in mismatch repair and BLM deficient HCT116 BLM-/- cells.