Salmonella entericaEnteritidis is the second most common serovar associated with invasive non-typhoidalSalmonella(iNTS) disease in sub-Saharan Africa. Previously, genomic and phylogenetic characterization ofS.entericaEnteritidis isolates from the human bloodstream led to the discovery of the Central/Eastern African clade (CEAC) and West African clade, which were distinct from the gastroenteritis-associated global epidemic clade (GEC). The AfricanS.entericaEnteritidis clades have unique genetic signatures that include genomic degradation, novel prophage repertoires and multi-drug resistance, but the molecular basis for the enhanced propensity of AfricanS.entericaEnteritidis to cause bloodstream infection is poorly understood. We used transposon insertion sequencing (TIS) to identify the genetic determinants of the GEC representative strain P125109 and the CEAC representative strain D7795 for growth in threein vitroconditions (LB or minimal NonSPI2 and InSPI2 growth media), and for survival and replication in RAW 264.7 murine macrophages. We identified 207in vitro-required genes that were common to bothS.entericaEnteritidis strains and also required byS.entericaTyphimurium,S.entericaTyphi andEscherichia coli, and 63 genes that were only required by individualS.entericaEnteritidis strains. Similar types of genes were required by both P125109 and D7795 for optimal growth in particular media. Screening the transposon libraries during macrophage infection identified 177 P125109 and 201 D7795 genes that contribute to bacterial survival and replication in mammalian cells. The majority of these genes have proven roles inSalmonellavirulence. Our analysis uncovered candidate strain-specific macrophage fitness genes that could encode novelSalmonellavirulence factors.