Listeria monocytogenes has emerged as a food safety concern for a number of produce commodities. While L. monocytogenes contamination can occur throughout the supply chain, contamination from the packinghouse environment represents a particular challenge and has been linked to recalls. This study aimed to investigate the prevalence, persistence, and diversity of Listeria monocytogenes (LM) and other Listeria species (LS) in produce packinghouses. A longitudinal study was performed in 11 packinghouses (commodities included micro-green, peach, apple, tomato, broccoli, cauliflower, and cucumber) in three US states. In each packinghouse, 34 to 46 sites representing zones 2-4 were selected and swabbed. Packinghouses were visited 4 times and samples were processed for Listeria by US Food and Drug Administration's Bacteriological Analytical Manual methods. Presumptive Listeria-positive isolates were confirmed by PCR. Species and allelic type (AT) were identified by sigB sequencing. Among the 1,584 samples tested, 3.2%, 2.7%, and 0.6% of the samples were positive for LM, LS, and both LM and LS, respectively. Five different species of Listeria were identified with L. monocytogenes being the most prevalent species. A high AT diversity (0.95 Simpson's Diversity Index) was observed amongst Listeria isolates. There were 15 instances of Listeria repeated isolation (site testing positive ≥2 times). Upon analysis of subtype data, only 3 sites tested positive for the same Listeria AT >2 times. Data showed in this longitudinal study that Listeria prevalence and persistence in packinghouses was low (e.g., <4% prevalence). Therefore, sanitation program development and implementation in packinghouses are critical to limit Listeria harborage and residence.