Accurate diagnostic tests are essential for the correct identification of etiologic agents causing sepsis. Conventional microbiology cultures are time consuming and may even yield negative results in many cases of septic shock. In this manner, molecular-based technologies are emerging as promising tests for use into routine clinical laboratories. In this review, we discuss current available molecular methods for bacteremia diagnosis in adult and pediatric patients with suspected or confirmed sepsis. Results of studies using polymerase chain reaction, real-time polymerase chain reaction, and complementary DNA/oligonucleotide microarrays are described and discussed into the current scenario. These new methodologies are able to detect even small amounts of bacterial DNA directly from blood specimens and show increased sensitivity and specificity for detecting many infectious agents associated with sepsis. Despite some limitations presented by nucleic acid-based techniques, these genotypic tests can be useful along with traditional microbiology diagnostics.