The Accelerate PhenoTM system (APS, Accelerate Diagnostics) is approved for the rapid identification and phenotypic antimicrobial susceptibility testing (AST) of microorganisms grown from positive blood cultures inoculated with blood from septic patients. We evaluated the performance for identification and AST from positive blood culture bottles inoculated with primary sterile non-blood specimens from patients with suspected severe infections. One hundred positive blood culture bottles with primary sterile specimens (63 cerebrospinal fluids, 16 ascites, 7 pleural fluids, 4 vitreous fluids, 5 joint aspirates and 5 other aspirates) from 100 patients were included. Pathogen identification was in agreement with conventional methods for 72 of 100 cultures (72%) and for 81 of 112 (72%) pathogens when considering all pathogens, for 72 of 92 (78%) cultures and 81 of 104 (78%) pathogens when considering on-panel pathogens only. Eight of 31 isolates (26%) not identified by APS were pathogens not included in the APS panel. APS and conventional methods accordingly identified all pathogens from two of nine polymicrobial cultures (22%). APS generated antimicrobial resistance results for 57 pathogens of 57 cultures. The overall category agreement between APS and culture based AST was 91.2%, the rates for minor errors 6.9%, major 1.7% and very major errors 0.2%. APS may accelerate pathogen identification and phenotypic AST from positive blood culture bottles inoculated with primary sterile specimens from patients with serious infections, especially for hospitals without on-site microbiology laboratory. However, the inclusion of non-blood specimens with a high likelihood of polymicrobial infections may result in an inferior performance.