Deciphering the origin of invasive plant species, whether or not there have been multiple introductions, and genetic differentiations between invasive and native ranges is crucial in testing hypotheses underlying biological invasions. Here, we applied traditional population genetic analyses to unravel the phylogeographical relationships among invasive (North American) and native (North African, Mediterranean region, and Eurasian) range populations of Brassica nigra using chloroplast DNA. We sequenced chloroplast DNA intron (trnF–trnL) for 284 individuals representing 36 native and 15 invasive range populations of B. nigra. Thirty-two haplotypes were found over the whole data set. A similarity between the invasive range and native range populations in genetic diversity combined with results from analyses of molecular variance and gene genealogies suggest that invasive B. nigra populations were introduced from multiple sources in the native range. More generally, this study adds to the growing body of data on the genetic patterns involved in biological invasions that is crucial to our understanding of the evolutionary trajectories of invasive populations.