Background Invasive infections with non-typhoidal Salmonella (NTS) lead to bacteremia in children and adults and are an important cause of illness in Africa; however, few data on the burden of NTS bacteremia are available. We sought to determine the burden of invasive NTS disease in a rural and urban setting in Kenya. Methods We conducted the study in a population-based surveillance platform in a rural setting in western Kenya (Lwak), and an informal urban settlement in Nairobi (Kibera) from 2009 to 2014. We obtained blood culture specimens from participants presenting with acute lower respiratory tract illness, or acute febrile illness to a designated outpatient facility in each site, or any hospital admission for a potentially infectious cause (rural site only). Incidence was calculated using a defined catchment population and adjusting for specimen collection and healthcare seeking practices. Results A total of 12,683 and 9,524 blood cultures were analyzed from Lwak and Kibera, respectively. Of these, 428 (3.4%) and 533 (5.6%) grew a pathogen; among those 208 (48.6%) and 70 (13.1%) were positive for NTS in Lwak and Kibera, respectively. Overall, the adjusted incidence of invasive NTS disease was higher in Lwak (839.4 per 100,000 person-years observation [pyo]) compared with Kibera (202.5 per 100,000 pyo). The highest adjusted incidences were observed in children <5 years of age (Lwak 3,914.3 per 100,000 pyo and Kibera 997.9 per 100,000 pyo). In Lwak the highest adjusted annual incidence was 1,927.3 per 100,000 pyo (in 2010) and in Kibera 220.5 per 100,000 pyo (in 2011); the lowest incidences were 303.3 and 62.5 per 100,000 pyo respectively (in 2012). In both sites, invasive NTS disease incidence generally declined over the study period. Conclusion We observed an extremely high burden of invasive NTS disease in a rural area of Kenya and a lesser, but still substantial, burden in an urban slum. Although the incidences in both sites declined during the study period, invasive NTS infections remain an important cause of morbidity in these settings, particularly among children < 5 years old.