is an animal and zoonotic pathogen of worldwide importance. serovars that differ in their host and tissue tropisms exist. Cattle are an important reservoir of human nontyphoidal salmonellosis, and contaminated bovine peripheral lymph nodes enter the food chain via ground beef. The relative abilities of different serovars to survive within the bovine lymphatic system are poorly understood and constrain the development of control strategies. This problem was addressed by developing a massively parallel whole-genome sequencing method to study mixed-serovar infections serovars differ genetically by naturally occurring single nucleotide polymorphisms (SNPs) in certain genes. It was hypothesized that these SNPs could be used as markers to simultaneously identify serovars in mixed populations and quantify the abundance of each member in a population. The performance of the method was validated using simulated pools containing up to 11 serovars in various proportions. It was then applied to study serovar survival in cattle challenged orally with the same 11 serovars. All the serovars successfully colonized the bovine lymphatic system, including the peripheral lymph nodes, and thus pose similar risks of zoonosis. This method enables the fates of multiple genetically unmodified strains to be evaluated simultaneously in a single animal. It could be useful in reducing the number of animals required to study mixed-strain infections and in testing the cross-protective efficacy of vaccines and treatments. It also has the potential to be applied to diverse bacterial species which possess shared but polymorphic alleles. While some serovars are more frequently isolated from lymph nodes rather than the feces and environment of cattle, the relative abilities of serovars to survive within the lymphatic system of cattle remain ill defined. A sequencing-based method which used available information from sequenced genomes to study the dynamics of mixed-serovar infections was developed. The main advantages of the method include the simultaneous identification and quantification of multiple strains without any genetic modification and minimal animal use. This approach could be used in vaccination trials or in epidemiological surveys where an understanding of the dynamics of closely related strains of a pathogen in mixed populations could inform the prediction of zoonotic risk and the development of intervention strategies.