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Spiral nematodes (<i>Helicotylenchus</i> spp.) are polyphagous parasitic species exhibiting a broad host range and geographical distribution. However, their diversity in the cultivated regions of southern Alberta remains understudied. Hence, we conducted a comprehensive survey of the region’s arable lands for the presence of spiral nematodes and revealed two <i>Helicotylenchus</i> species, <i>H. crassatus</i> and <i>H. oscephalus</i>. <i>H. crassatus</i> consisted of two distinct morphotypes: one morphotype had a conoid tail with slight ventral projection on the distal end, whereas the other had a broadly rounded tail. This study presents the first documentation of <i>H. crassatus</i> and <i>H. oscephalus</i> from southern Alberta, Canada. Molecular characterization was based on the partial 18S rRNA, the D2–D3 of 28S rRNA, ITS rRNA, and <i>COI</i> gene sequences, complemented by detailed morphological studies using scanning electron microscopy. In this work, <i>Helicotylenchus</i> species were often co-detected with root lesion nematodes, which made the evaluation of their role in crop damage more difficult. To meet the requirements for threshold and pathogenicity assessments, we introduced both spiral nematode species to sterile carrot disks and evaluated the feasibility of their multiplication and mass production in vitro. The present findings expand the taxonomic records of <i>Helicotylenchus</i> spp. and improve diagnostics of these morphologically similar species. Furthermore, our in vitro culture technique will provide a reliable source of the initial inoculum for future plant–nematode interaction studies.