Phylogenetic analyses of the 18S small-subunit (SSU) rRNA and Cryptosporidium oocyst wall protein (COWP) genes indicate that multiple unique Cryptosporidium isolates are shed by separate species of Spermophilus ground squirrels (S. beecheyi, S. beldingi, and S. lateralis) throughout California, despite some squirrel populations' being geographically isolated. The data support our assertion that Spermophilus squirrels shed novel Cryptosporidium species.