The emerging field of synthetic genetics provides an opportunity to explore the structural and functional properties of synthetic genetic polymers by in vitro selection. Limiting this process, however, is the availability of enzymes that allow for the synthesis and propagation of genetic information present in unnatural nucleic acid sequences. Here, we report the development of a transcription and reverse-transcription system that can replicate unnatural genetic polymers composed of threose nucleic acids (TNA). TNA is a potential progenitor of RNA in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. Using commercial polymerases that recognize TNA, we demonstrate that an unbiased three-letter and two different biased four-letter genetic alphabets replicate in vitro with high efficiency and high overall fidelity. We validated the replication system by performing one cycle of transcription, selection, reverse transcription, and amplification on a library of 10(14) DNA templates and observed similar to 380-fold enrichment after one round of selection for a biotinylated template. We further show that TNA polymers are stable to enzymes that degrade DNA and RNA. These results provide the methodology needed to evolve biologically stable aptamers and enzymes for exobiology and molecular medicine.