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The Neotropics of South America represent one of the most diverse assemblages of freshwater organisms in the world. The geologic and ecological changes that have occurred throughout the Amazon and Orinoco River basins have resulted in the two most diverse rivers in the world producing very heterogeneous environments potentially influencing current population structure of Neotropical species. To investigate specific ecological and geological events influencing populations in these regions, I explore population structure and demographic histories of two species from the genus Cichla with wide distributions among the Amazon and Orinoco. First, I describe the methods used to isolate and characterize microsatellite loci for this genus. Using these microsatellite data as well as mitochondrial DNA sequences, I study the population structures for C. temensis, a species found in the Orinoco and Amazon spanning the Casiquiare River (a natural hydrogeologic corridor connecting the two rivers), and C. monoculus, which is the most widely distributed species in this genus found throughout the Amazon and the majority of its tributaries. The most significant results from my study show that the current population distribution of C. temensis is likely a result of population expansion across the Casiquare River and the contrasting water types found in the Orinoco and Amazon Rivers seem to be limiting gene flow to the immediate boundaries of the Casiquiare River. The analysis of C. monoculus shows strong population structure not reflective of geologic events, specifically the breaching of the Purus Arch. However, the different water types seem to be the most likely factor influencing population structure. While the analysis for both species has indicated that geologic histories of these river basins may have influenced their current population structure, ecological variation seems to have a more dramatic effect on current gene flow across their wide distributions.