The juxtaposition and functional relationship of the sperm storage organs in Anastrepha ludens (Loew) is described. The spermatheca squash technique has been used to determine mated status in tephritid fruit flies, and thus as a measure of compatibility and coverage for sterile insect technique programs. Female A. ludens have four seminal storage organs: three spermathecae and a ventral receptacle. By interrupting coitus at set intervals, it is shown that the ventral receptacle fills first and after 15–30 min the spermathecae begin to fill. Among wild captured flies, all mature (gravid) females had semen in the ventral receptacle, but only 40 – 60% had sperm in at least one spermatheca. Therefore, examination of the ventral receptacle rather than the spermathecae alone is the more reliable method for determining mated status.