Late-onset sepsis (LOS) is a common and life-threatening problem in neonates, particularly among infants requiring intensive care. LOS can be caused by a variety of organisms; gram-positive organisms are most common and include staphylococcal species, group B or A Streptococcus, and Enterococcus. A wide range of gram-negative and fungal organisms, including Candida, are also common causes of LOS. A large proportion of LOS is due to horizontal transfer of pathogens within the intensive care nursery or indwelling medical devices, but cases may also be due to colonizing organisms from the infant’s mucosal membranes or gut. This chapter discusses the diagnostic and treatment approach to late-onset sepsis, including focal infections such as urinary tract infection, pneumonia, skin and soft tissue infection, and osteoarticular infection.