This study sought to determine whether a change in twinning prevalence was associated with mandatory folic acid fortification. As of January 1998, it became mandatory in the United States that grain products be fortified with folic acid. The effectiveness of this fortification for neural tube defects has been explored, but other possible implications of fortification has not. We computed the prevalence of twinning among more than 2.5 million California births occurring from 1990 to 1999. The prevalence of twin births in California increased over the last decade with increases observed among most maternal race/ethnic, age, and parity groupings. Unadjusted prevalence measures did not reveal substantial increases in twinning prevalence among these groupings after the fortification period relative to the period immediately preceding it. Analyses that simultaneously adjusted for age, parity, race/ethnicity, sexes of twin pair (as a measure of zygosity), year of birth, and fortification period also did not reveal an association between fortification and twinning. Our results do not suggest a change in twinning prevalence associated with folic acid fortification of the US food supply among women delivering in California.