The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX® system and RAPID-B® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX® system and RAPID-B® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B® flow cytometer and USDA-FSIS reference method. The RAPID-B® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B® flow cytometer based on whole cell detection generated similar results as BAX® system therefore the RAPID-B® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products. •We compared the non-O157 STEC detection sensitivity between BAX® system and RAPID-B® flow cytometer.•The RAPID-B® flow cytometer exhibited similar results as BAX® system.•The RAPID-B® flow cytometer is a rapid detection method for non-O157 STEC in beef products.