Brinjal little leaf (BLL) disease, caused by phytoplasmas, leads to significant yield losses, but its pathogenic mechanisms remain poorly understood. This study characterizes SAP54LP/S54LP (SAP54-like protein of BLL), an effector protein associated with the 16SrVI-D phytoplasma strain linked to BLL in Tamil Nadu, India. Molecular analyses, including nested PCR and virtual RFLP, confirmed the phytoplasma's classification within the 16SrVI group. The S54LP effector gene was amplified from infected brinjal, confirming its presence. Sequence comparisons showed that S54LP was 100 % identical to homologs in the 16SrVI-A group and 98 % identical to those in the 16SrIB group (phyl1 and S54LP), indicating group-specific divergence. Comparative analysis identified 13 single nucleotide polymorphisms (SNPs), 69 % of which led to nonsynonymous substitutions in the mature protein, potentially affecting host interactions. Haplotype network analysis revealed a globally distributed Hap_6 lineage and region-specific haplotypes, suggesting localized evolutionary dynamics. Evolutionary analysis indicated strong purifying selection on the coding sequence (Ka/Ks ≈ 0.3), reflecting the conservation of functional domains essential for pathogenicity. Conversely, positive selection was detected in the signal peptide region, implying adaptive evolution related to secretion and host targeting. Additionally, AT-rich codon usage at specific sites highlights multiple layers of evolutionary constraints shaping S54LP. Overall, these findings establish S54LP as a model for studying host-phytoplasma coevolution, providing insights into effector biology and informing strategies to manage phytoplasma diseases in crops.