Esophageal glands of plant-parasitic nematodes are highly specialized cells whose gene expression products include secreted effector proteins, which govern nematode parasitism of host plants. Therefore, elucidating the transcriptomes of esophageal glands with the goal of identifying nematode effectors is a promising avenue to understanding nematode parasitism and its evolutionary origins as well as to devising nematode control strategies. We have developed a method to separate and isolate individual esophageal gland cells from multiple species of plant-parasitic nematodes while preserving RNA quality. We have used such isolated gland cells for transcriptome analysis via high-throughput DNA sequencing. This method relies on the differential histochemical staining of the gland cells after homogenization of phytonematode tissues. Total RNA was extracted from whole gland cells isolated from eight different plant-parasitic nematode species. To validate this approach, the isolated RNA from three plant-parasitic nematode species-Globodera rostochiensis, Pratylenchus penetrans, and Radopholus similis-was amplified, gel purified, and used for 454 sequencing. We obtained 456,801 total reads with an average read length of 409 bp. Sequence analyses revealed the presence of homologs of previously known nematode effectors in these libraries, thus validating our approach. These data provide compelling evidence that this technical advance can be used to relatively easily and expediently discover effector repertoires of plant-parasitic nematodes.