Investigations aimed at defining the 3-D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. High-throughput sequencing has only recently made extensive genomic mapping of eccDNA sequences possible and had yet to be applied using a rigorous approach that distinguishes ascertainment bias from true enrichment. Here, we define eccDNA distribution, utilizing a set of unbiased topology-dependent approaches for enrichment and characterization. We use parallel biophysical, enzymatic, and informatic approaches to obtain a comprehensive profiling of eccDNA in C. elegans and in three human cell types, where eccDNAs were previously uncharacterized. We also provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture in which endogenous genomic DNA circles are present in normal physiological DNA metabolism, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.