During in vitro broth culture, bacterial gene expression is typically dominated by highly expressed factors involved in protein biosynthesis, maturation, and folding, but it is unclear if this also applies to conditions in natural environments. Here, we used a promoter trap strategy with an unstable green fluorescent protein reporter that can be detected in infected mouse tissues to identify 21 Salmonella enterica promoters with high levels of activity in a mouse enteritis model. We then measured the activities of these and 31 previously identified Salmonella promoters in both the enteritis and a murine typhoid fever model. Surprisingly, the data reveal that instead of protein biosynthesis genes, disease-specific genes such as Salmonella pathogenicity island 1 (SPI-1)-associated genes and genes involved in anaerobic respiration (enteritis) or SPI-2-associated genes and genes of the PhoP regulon (typhoid fever), respectively, dominate Salmonella in vivo gene expression. The overall functional profile of highly expressed genes suggests a marked shift in major transcriptional activities to nutrient utilization during enteritis or to fighting against the host during typhoid fever. The large proportion of known and novel essential virulence factors among the identified genes suggests that high expression levels during infection may correlate with functional relevance.