Technical Abstract: After the 2004 discovery of the Bemisia tabaci Q biotype in the U.S., there was an urgent need to determine its distribution. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in Florida, with the cooperation of growers and state agencies, to monitor the introduction and distribution of both the B and Q biotypes. The biotype status of submitted B. tabaci samples was determined by PCR amplification and sequencing of a 700 - 800 bp mitochondrial cytochrome oxidase I small subunit (mtCOI) gene fragment, PCR amplification and size determination of 2 unique microsatellite markers and esterase zymogram analysis. One hundred and eighty collections were sampled from 23 counties. Of these samples, 58 percent were from vegetables, 37 percent were from ornamentals and 5 percent were from peanuts, alfalfa and weeds. Eighteen percent of all collections were found to be the Q biotype that came from greenhouse grown ornamental and herbs located in six counties. Sequence comparison of the mtCOI gene identified three separate haplotypes within Florida that were defined as Q1, Q2 and Q3. Haplotypes could be used to associate populations known to be related by grower and plant type. For example, collections from five counties were made on hibiscus linked to the same grower and all samples contained only the Q1 haplotype. Other populations contained a mix of the Q2 and Q3 haplotypes supporting the conclusion that the Q biotype must have entered Florida through at least two separate introductions. Our data also show that two microsatellite markers are cost effective diagnostic alternatives for biotype ID with 100 percent concurrence with mtCOI sequence data.