The host-restricted Lancefield group C pathogen Streptococcus equi infects the lymph nodes in the head and neck of horses, forming abscesses which may become so large that the horse’s airways are obstructed, lending to the disease’s name, strangles. We have developed a barcoded TraDIS system that identifies coding sequences (CDSs) required for the virulence of S. equi in a susceptible natural host. Twelve Welsh mountain ponies were each infected with two of three barcoded ISS1 libraries each containing 33,000-50,000 unique mutants. These libraries contained, on average, an ISS1 transposition every 56 bp, or an average of 19 unique mutants per CDS. Ponies were euthanased on developing early clinical signs, typically pyrexia and preference for haylage and water over dry-pelleted food. Viable mutants were recovered from the abscess material of 24 retropharyngeal and 14 submandibular lymph nodes. Sequencing across ISS1-genome junctions identified 492 CDSs, which were non-essential in vitro, but were significantly depleted in the abscess pools. Depleted mutants included insertions in lgt or prtM, which are known to reduce the ability of S. equi to cause disease in ponies. Significant decreases in fitness were also measured in sagA, csrS and SEQ_2190 (surface-anchored protein). Mutants in a further 239 CDSs, including those encoding 19 putative membrane proteins, were significantly enriched in the abscess pools. Our data provide an unprecedented insight into the mechanisms employed by S. equi to cause disease in the natural host. Our findings are likely to also shed light on pathways important for virulence in other streptococci.