Highway-associated, lentic freshwater habitats are ubiquitous throughout the landscape of the southern USA and support diverse assemblages of aquatic insects. However, because of their generally small size, dynamic hydro-period, and disturbed nature, these habitats are often overlooked by naturalists, and subsequently faunal surveys are rare. This work addressed this knowledge gap by specifically surveying for a common and widespread group of beetles within these habitats, predaceous diving beetles (Coleoptera: Dytiscidae). Semi-permanent and ephemeral freshwater habitats associated with two major state highways and one interstate highway were sampled in early and late summer of 2009 within a 40-km radius of Hattiesburg, MS. Among 15 tire ruts, 18 ditches, and 30 ponds, we collected 861 adult and 545 larval dytiscids representing 18 different species. Our findings suggest that highway-associated, freshwater habitats may act as important habitat and dispersal “stepping-stones” for certain species of dytiscids in the landscape and likely play a part in maintaining the metapopulations and regional biodiversity of these taxa.