Aims To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates. Methods and Results Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (mu l) of each culture suspension (1x 108 CFU ml-1) were added to 20 mu l of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates. Conclusions Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities =97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains. Significance and Impact of the Study This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.