Spatial working memory (SWM) involves both simultaneous and sequential encoding, but the differences in their neural correlates are unclear. We investigated the differences in prefrontal cortex activity related to these SWM encoding types. We also examined the patterns of brain activity influencing individual visuospatial abilities (VSA). We conducted SWM tasks with two different conditions, sequential and simultaneous encoding, and examined hemodynamic activity in 39 healthy adults using nearinfrared spectroscopy. The bilateral dorsolateral prefrontal cortex was activated more strongly in the sequential condition compared with the simultaneous condition. This suggests that prefrontal cortex activity underlying SWM is modulated by the type of encoding. We also found that individuals with high VSA showed weaker activation in the right-dorsolateral prefrontal cortex compared with those with lower VSA during the simultaneous condition. This hypoactivation is thought to reflect neural efficiency in the individuals with high ability. These findings are expected to lead to a better understanding of neural substrates for SWM. Copyright (C) 2017 Wolters Kluwer Health, Inc. All rights reserved.