Bovine peripheral lymph nodes (LNs), including subiliac LNs, have been identified as a potential source of human exposure to Salmonella enterica, when adipose trim containing these nodes is incorporated into ground beef. In order to gain a better understanding of the burden of S. enterica in peripheral LNs of feedlot and cull cattle, a cross-sectional study was undertaken in which 3327 subiliac LNs were collected from cattle at harvest in seven plants, located in three geographically distinct regions of the United States. Samples were collected in three seasons: Fall 2010, Winter/Spring 2011, and Summer/Fall 2011. A convenience sample of 76 LNs per day, 2 days per season (approximately 1 month apart), was collected per plant, from carcasses held in the cooler for no less than 24 h. Every 10th carcass half on a rail was sampled, in an attempt to avoid oversampling any single cohort of cattle. Median point estimates of S. enterica contamination were generally low (1.3%); however, median Salmonella prevalence was found to be greater in subiliac LNs of feedlot cattle (11.8%) compared to those of cull cattle (0.65%). Enumeration analysis of a subset of 618 feedlot cattle LNs showed that 67% of those harboring S. enterica (97 of 144) did so at concentrations ranging from <0.1 to 1.8 log10 CFU/g, while 33% carried a higher burden of S. enterica, with levels ranging from 1.9 to >3.8 log10 CFU/g. Serotyping of S. enterica isolated identified 24 serotypes, with the majority being Montevideo (44.0%) and Anatum (24.8%). Antimicrobial susceptibility phenotypes were determined for all isolates, and the majority (86.1%) were pansusceptible; however, multidrug-resistant isolates (8.3%) were also occasionally observed. As Salmonella contained within LNs are protected from carcass interventions, research is needed to define opportunities for mitigating the risk of Salmonella contamination in LNs of apparently healthy cattle.