This study presents the assembly and comparative genomic analysis of luminous
Photobacterium
strains isolated from the light organs of 12 fish species using Oxford Nanopore Technologies (ONT) sequencing. The majority of assemblies achieved chromosome-level continuity, consisting of one large (>3 Mbp) and one small (~1.5 Mbp) contig, with near complete BUSCO scores along with varying plasmid sequences. Leveraging this dataset, this study significantly expanded the available genomes for
P. leiognathi
and its subspecies P. ‘mandapamensis’, enabling a comparative genomic analysis between the two lineages. An analysis of the large and small chromosomes unveiled distinct patterns of core and accessory genes, with a larger fraction of the core genes residing on the large chromosome, supporting the hypothesis of secondary chromosome evolution from megaplasmids in Vibrionaceae. In addition, we discovered a proposed new species, Photobacterium acropomis sp. nov., isolated from an acropomatid host, with an average nucleotide identify (ANI) of 93 % compared to the
P. leiognathi
and P. ‘mandapamensis’ strains. A comparison of the
P. leiognathi
and P. ‘mandapamensis’ lineages revealed minimal differences in gene content, yet highlighted the former’s larger genome size and potential for horizontal gene transfer. An investigation of the lux-rib operon, responsible for light production, indicated congruence between the presence of luxF and host family, challenging its role in differentiating P. ‘mandapamensis’ from
P. leiognathi
. Further insights were derived from the identification of metabolic differences, such as the presence of the NADH:quinone oxidoreductase respiratory complex I in
P. leiognathi
as well as variations in the type II secretion system (T2S) genes between the lineages, potentially impacting protein secretion and symbiosis. In summary, this study advances our understanding of
Photobacterium
genome evolution, highlighting subtle differences between closely related lineages, specifically
P. leiognathi
and P. ‘mandapamensis’. These findings highlight the benefit of long read sequencing for bacterial genome assembly and pangenome analysis and provide a foundation for exploring early bacterial speciation processes of these facultative light organ symbionts.