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The average nucleotide identity (ANI) metric has become the gold standard for prokaryotic species delineation in the genomics era. The most popular ANI algorithms are available as command-line tools and/or web applications, making it inconvenient to incorporate them into bioinformatic workflows, which utilize the popular Python programming language. Here, we present PyOrthoANI, PyFastANI, and Pyskani, Python libraries for three popular ANI computation methods. ANI values produced by PyOrthoANI, PyFastANI, and Pyskani are virtually identical to those produced by OrthoANI, FastANI, and skani, respectively (adjusted R2>0.999). Compared to OrthoANI, PyOrthoANI is, on average, 3× faster per genome, while PyFastANI has multithreading support for single queries. All three libraries integrate seamlessly with BioPython, making it easy and convenient to use, compare, and benchmark popular ANI algorithms within Python-based bioinformatic workflows, software programs, and notebooks. Each library is available as part of the Python Package Index repository under the open-source MIT license, with source code available via GitHub (PyOrthoANI, https://github.com/althonos/orthoani; PyFastANI, https://github.com/althonos/pyfastani; Pyskani, https://github.com/althonos/pyskani).