Hop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding genome evolution in Humulus represents a considerable challenge, requiring additional resources, including integrated genome maps. In order to facilitate cytogenetic investigations into the transmission genetics of hop, we report here the identification and characterization of 17 new and distinct tandem repeat sequence families. A tandem repeat discovery pipeline was developed using kmer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. We produced oligonucleotide FISH probes from conserved regions of HuluTR120 andHulTR225 and demonstrated their utility to stain meiotic chromosomes from wild hop, var.neomexicanus. The HuluTR225 FISH probe hybridized to several loci per nucleus and exhibited irregular, non-Mendelian transmission in male meiocytes of wild hop. Collectively, these tandem repeat sequence families not only represent unique and valuable new cytogenetic reagents but also have the capacity to inform genome assembly efforts and support comparative genomic analyses.