The US Environmental Protection Agency (USEPA) conducted a National Lakes Assessment (NLA) in the conterminous USA in 2007 as part of a national assessment of aquatic resources. The EPA used the National Hydrography Dataset (NHD) as the basis for the sample frame for the NLA. The target population was all lakes >4 ha, excluding the Laurentian Great Lakes and the Great Salt Lake. An unequal probability survey design was used to select 4472 candidate lakes for potential sampling. The unequal selection depended on 5 lake area classes and 9 aggregated Omernik level III ecoregions. In all, 2034 candidate lakes were evaluated for inclusion in the target population, and 1309 lakes (representing ∼68,000 lakes in the sample frame) met the criteria. A total of 1028 lakes (of 1309) were sampled and represented ∼50,000 lakes. The remaining lakes (231, representing ∼18,000 lakes) could not be sampled because of access denial or physical inaccessibility. The target population included natural (41 ± 2% [SE]) and man-made lakes (59 ± 2%). All target lakes in the Southern Appalachian region and >90% of the target population in the Southern Plains and Xeric regions were man-made. In the Upper Midwest region, 97 ± 1% of the target population were natural lakes. Small lakes (4–10 ha) made up 47 ± 2% of the target population, and lakes >50 ha made up ∼15% of the target population. The results raise 2 issues that have implications for current and future NLA projects: 1) the cost and effort required to identify lake features in the sample frame that do not meet the criteria for inclusion in the target population (∼50% in NLA 2007), and 2) the potential for biased estimates of the size and condition of the target population caused by lakes that cannot be sampled. Future NLA efforts involve refining the survey design to include smaller lakes and resampling lakes from previous NLAs. We offer approaches for addressing both issues, including use of a high-resolution version of NHD as the basis for developing the NLA sample frame. Developing a master sample frame of lakes would provide a consistent basis of lake numbers (or surface area) from which to estimate extent or assess ecological condition.