Pratylenchus penetrans is one of the most important species among root lesion nematodes (RLNs) due to the detrimental and economic impact that it causes in a wide range of crops. Similar to other plant-parasitic nematodes (PPNs), P. penetrans harbors a significant number of secreted proteins that play key roles during parasitism. Here we combined spatially and temporally resolved next generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effectors within the esophageal glands of P. penetrans by in situ hybridization. These comprised homologues of known effectors of other PPNs with diverse putative functions, as well as novel pioneer effectors specific to RLNs. It is noteworthy that five of the pioneer effectors encode extremely proline-rich proteins. We then combined in situ localization of effectors with available genomic data to identify a non-coding motif enriched in promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. Expression profiling analyses of a subset of candidate effectors confirmed their expression during plant infection. Our current results provide the most comprehensive panel of effectors found for RLNs. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the mode of parasitism of this nematode and offers useful suggestions regarding the potential use of P. penetrans-specific target effector genes to control this important pathogen. This article is protected by copyright. All rights reserved.