We have adapted Sternberg's context-recall task to investigate the neural mechanisms of encoding serial order information in working memory, in 2 male rhesus monkeys. We recorded from primary motor, premotor, and dorsolateral prefrontal cortex while the monkeys performed the task. In each cortical area, most neurons displayed marked modulation of activity during the list presentation period of the task, whereas the serial order of the stimuli needed to be encoded in working memory. The activity of many neurons changed in a consistent manner over the course of the list presentation period, without regard to the location of the stimuli presented. Remarkably, these neurons encoded serial position information in a relative (rather than absolute) manner across different list lengths. In addition, many neurons showed activity related to both location and serial position, in the form of an interaction effect. Surprisingly, the activity of these neurons was often modulated by the location of stimuli presented before the epoch in which the activity changes occurred. In motor and premotor areas, a large proportion of neurons with list presentation activity also showed direction-related activity during the response phase, whereas in prefrontal cortex most cells showed only list presentation effects. These results show that many neurons had a heterogeneous functionality by representing distinct task variables at different periods of the task. Finally, potential confounds could not account for the effects observed. For these reasons, we conclude that these neurons were indeed participating in sequence encoding in working memory.SIGNIFICANCE STATEMENT Traditionally, primary motor, premotor, and prefrontal areas have been considered to be mainly engaged in motor output, visuomotor transformation, and higher cognitive functions, respectively. Here we show that neurons in all three cortical regions participate in the encoding of a sequence of spatial stimuli in working memory. Furthermore, a central question in cognitive neuroscience has been the manner in which the position of an item within a sequence is encoded in the brain. Our findings provide direct neurophysiological support for a specific hypothesis from cognitive psychology: that of relative coding of serial order.