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Background: Invasive non-typhoidal Salmonella (iNTS) serovars S. Typhimurium and S. Enteritidis are major etiologic agents of invasive bacterial disease among infants and young children in sub-Saharan Africa, including in Mali. Early studies of iNTS serovars in several countries indicated that S. Typhimurium was more prevalent than S. Enteritidis, including in Mali before 2008. We investigated genomic and associated phenotypic changes associated with an increase in the relative proportion of iNTS caused by S. Enteritidis versus S. Typhimurium in Bamako, Mali, during the period 2002–2012. Methodology/Principal findings: Comparative genomics studies identified homologs of tetracycline resistance and arsenic utilization genes that were associated with the temporal shift of serovars causing iNTS shift, along with several hypothetical proteins. These findings, validated through PCR screening and phenotypic assays, provide initial steps towards characterizing the genomic changes consequent to unknown evolutionary pressures associated with the shift in serovar prevalence. Conclusions/Significance: This work identified a shift to S. Enteritidis from the more classic S. Typhimurium, associated with iNTS in Bamako, Mali, during the period 2002–2012. This type of shift in underlying iNTS pathogens are of great importance to pediatric public health in endemic regions of sub-Saharan Africa. Additionally, this work demonstrates the utility of combining epidemiologic data, whole genome sequencing, and functional characterization in the laboratory to identify and characterize genomic changes in the isolates that may be involved with the observed shift in circulating iNTS agents. [ABSTRACT FROM AUTHOR]