Symphylans are soil-dwelling arthropods that can cause significant damage to agricultural crops, particularly pineapple. Despite their economic importance, their taxonomy and biodiversity remain poorly understood in Colombia, and the Neotropics. Here the symphylan species associated with pineapple crops were investigated in Santander, Colombia, the country's largest pineapple-producing region. Symphylans were sampled from four commercial pineapple fields using baited pitfall traps. Morphological examination and DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene were used to identify the collected specimens. Six symphylan morphospecies were identified, including four Hanseniella and two Symphylella. The molecular analysis revealed four distinct genetic clades within the sequenced specimens. The integration of morphological and molecular data resolved initial taxonomic uncertainties, indicating that some previously separated morphospecies represent intraspecific morphological variation. Our results conclude that Hanseniella cf. unguiculata is the predominant species in pineapple crops, accounting for 95.9% of records. Additionally, two new species are described: Hanseniella chocoita sp. nov. and Hanseniella lebrijana sp. nov. A revised dichotomous key for the identification for Hanseniella species present in South America is also provided. This study provides valuable insights into the symphylan species inhabiting Colombian pineapple crops and emphasizes the need for further research to fully understand their diversity and evolutionary relationships.