A suspected glyphosate-resistant (R) junglerice population was collected from a glyphosate-R corn field near Durham in northern California where glyphosate had been applied at least twice a year for over 6 yr. Based on the amount of glyphosate required to reduce growth by 50% (ED50), the R population was 6.6 times more R than the susceptible (S) standard population. Based on the glyphosate concentration that inhibits EPSPS by 50% based on shikimate accumulation (I50) in leaf discs, R plants were four times more R than S plants. By 3 d after treatment with 0.42 kg ae ha−1 glyphosate, the S population had accumulated approximately five times more shikimate than the R population. No differences in [14C]-glyphosate uptake and translocation were detected between R and S plants. However, partial sequencing of the EPSPS gene revealed a mutation in R plants causing a proline to serine change at EPSPS position 106 (P106S). Our results reveal the first case of a P106S target site mutation associated with glyphosate resistance in junglerice.