This study was conducted to detect binary trait loci (BTLs) that influence guarding behavior of individual honey bees, Apis mellifera L., and to locate genetic markers that are associated with these BTLs on genetic maps derived from guard bees from two reciprocal backcross colonies. Samples of guards and control bees were taken from two backcross colonies derived from a defensive colony and a gentle colony. Amplified fragment length polymorphism (AFLP) markers were produced from DNA samples of guards. Two genetic maps were generated, one for each type of colony. A chi-square goodness-of-fit test was performed for each marker in the map to look for deviations from the 1:1 segregation of the markers. For those markers that significantly deviated from the 1:1 ratio in the guards, AFLPs were generated from two samples of control bees, one for each type of backcross. Those markers that showed a skewed segregation pattern in the guards but not in the controls were analyzed with a 2 × 2 chi-square to test for associations between the markers and the expression of the trait. Ten markers were associated with guarding behavior (five in each backcross). The 10 markers represented seven putative BTLs that influence honey bee guarding behavior. One of the BTLs represents a QTL that was previously detected in analysis of colony-level stinging response, others represent new loci specific to the behavior of individuals guarding the colony entrance.