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<div><i>Phytophthora nicotianae </i>is an Oomycete pathogen that causes diseases in ~225 plant genera, thus standing out among other <i>Phytophthora </i>species in its geographic distribution, host range and economic impact. In pineapple <i>(Annanas comosus)</i>, <i>P. nicotianae</i> causes heart rot disease, which significantly reduces crop yield due to plant mortality. Recent studies suggest that genotypes of this pathogen are host-specific to some extent; however, it is not clear how many host-specific lineages of the pathogen exist. The objective of this study was to determine the level of intra-specific genetic variation of <i>P. nicotianae</i> causing heart rot disease of pineapple in Ecuador. In addition, we aimed to compare the genotypes found on pineapple to lineages previously reported in citrus, tobacco and ornamentals. Thirty <i>P. nicotianae </i>colonies were collected from infected leaves of four farms during an outbreak in the 2016 rainy season in Santo Domingo province, Ecuador. We extracted DNA from single-spore isolates and confirmed them to be <i>P. nicotianae</i> by sequencing the cox2 gene. We genotyped the isolates using seven simple sequence repeat (SSR) loci. These loci revealed a single lineage that was distinct from those previously reported. These results support the hypothesis of host specialization of <i>P. nicotianae </i>in intensive farm systems and contribute to the understanding of the global population structure of this important pathogen.</div>