Whilst some bacteria have highly conserved genome structures (GSs), Salmonella frequently undergoes large-scale genomic rearrangements. These occur when large genome fragments shift position and/or orientation around the long-repeat sequences of ribosomal operons, producing unique GS variants. Such variation is linked to chronic carriage, as persistent isolates often display GS variation. Our previous research showed rearrangements, despite preserving total genetic content, can significantly alter phenotypes like growth rates, gene dosage, and metabolic function, with the most extreme changes coinciding with an imbalance in the length of the two replication forks (ori-ter imbalance). Here we investigate how genome rearrangements influence virulence and antibiotic resistance using isolates with GS variation but minimal/no SNPs. We analysed isolates from two sources: 1) derivatives from 12 clinical S. Typhi isolates maintained at ambient-temperature for over 3 years; 2) isolates from patients with chronic persistent S. Typhi or S. Agona infections. Multiplexed nanopore sequencing and the bioinformatic tool socru determined GSs, while short-read sequencing confirmed SNP absence. Changes in antibiotic susceptibility and virulence were assessed using disc diffusion assays and Galleria mellonella virulence model, respectively. Notably, some originally ciprofloxacin-resistant isolates displayed altered inhibition zones, suggesting sensitivity shifts due to genomic rearrangements. In S. Agona, fragment inversions leading to large ori-ter imbalances caused striking virulence changes, whereas inversions causing minor ori-ter disruption had little impact, suggesting these rearrangements may act as a virulence “on/off switch”. Our findings highlight a clear interplay between genome rearrangements, virulence and resistance – critical factors in management and control of Salmonella.