While it is of great significance for strawberry breeders to know the genetics of day-neutrality (DN), evidence for inheritance of the trait is still contradictory. A linkage mapping approach is being used to determine how many QTL regulate DN and the proportion of the variability explained by each. A preliminary genetic linkage map was constructed for 125 individuals of the day neutra× short day (SD) cross `Tribute' × `Honeoye' using single dose restriction fragments (SDRFs) of amplified fragment length polymorphic (AFLP) markers. Over 500 SDRFs from 55 AFLP primer combinations were used to build the map using the software tool Join Map 3.0 at a LOD score of 3.0. Single marker analysis using WinQTL cartographer software previously determined 27 SDRF markers to co-segregate with DN for 57 individuals of the mapping population phenotyped in the field for the years 2002 and 2003, indicating putative QTL for DN. These markers were included in the linkage analysis and seven of them mapped to five different linkage groups that may indicate the quantitative nature of the trait. For determining QTL and percentage of phenotype governed by each QTL, however, accurate phenotypic evaluation is critical. Therefore, controlled environment (growth chamber) studies were used to obtain flowering response data under SD and long day (LD) conditions with two day/night temperatures. This study was conducted for the entire mapping population (over 400 individuals) so that QTL detected can be confirmed by fine mapping the QTL regions. We will also test how robust the QTL detected are, by analyzing the same segregating population at six different field locations in the United States (California, Maryland, Michigan, Minnesota, New York, and Oregon) for their flowering response under SD and LD conditions.