The extreme drug resistance (EDR) assay has not been widely studied in the setting of non-metastatic breast cancer. We evaluated the feasibility of performing the assay in 144 primary breast tumor specimens from two institutions by determining the rate of successful tumor culture for assays, number of drugs evaluated per assay, and time from tumor biopsy to receipt of results. We also sought to determine factors that are associated with assay success. An exploratory analysis was performed to detect possible associations between estrogen receptor (ER), progesterone receptor (PR) and HER2/NEU over-expression and extreme drug resistance demonstrated by the assay for specific chemotherapeutic agents. Of 144 tumor specimens submitted, tumor was successfully cultured for assay in 101(70%) of cases. A median of five drugs was evaluated per assay (range 2-9). Results were obtained in a median of 8 days (range 2-29). Young age, high tumor grade, PR negativity, and higher tumor submission weight were predictive for a successful assay. EDR was observed in 7-15% of tumors to doxorubicin, cyclophosphamide, 5-fluorouracil (5FU) and mitoxantrone, but EDR to paclitaxel was observed in 35%. Extreme drug resistance to 5-FU was associated with negative ER and PR status. There was a trend toward association between EDR to paclitaxel and HER2/NEU over-expression. The EDR assay may be successfully performed in the majority of tumors, and assay results are available in a timely fashion such that adjuvant treatment drug selection could be guided by results. These results may be helpful for designing possible future trials that evaluate the assay's role in adjuvant chemotherapy selection.