AbstractTo explore the connection between chloroplast and coffee resistance factors, designated as S_H1 to S_H9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of S_H factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid “Híbrido de Timor”. Individual reads were also aligned on the C. arabica reference genome to relate S_H factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the S_H9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in S_H9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the S_H profile. This study introduces an unexplored strategy for identifying protein/genes associated with S_H factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.