Understanding sampling efficiency and movements of subadult Lake Sturgeon Acipenser fulvescens is necessary to facilitate population rehabilitation and recruitment monitoring in large systems with extensive riverine and lacustrine habitats. We used a variety of sampling methods to capture subadult Lake Sturgeon (i.e., fish between 75 and 130 cm TL that had not reached sexual maturity) and monitored their movements using radio telemetry in the lower Wolf River, a tributary to the Lake Winnebago system in Wisconsin. Our objectives were to determine whether (1) capture efficiency (expressed in terms of sampling time) of subadult Lake Sturgeon using multiple sampling methods was sufficient to justify within-river sampling as part of a basin-wide recruitment survey targeting subadults, (2) linear home ranges varied in relation to season or sex, and (3) subadult Lake Sturgeon remained in the lower Wolf River. From 2013 to 2014, 628 h of combined sampling effort that included gill nets, trotlines, electrofishing, and scuba capture was required to collect 18 subadult sturgeon, which were then implanted with radio transmitters and tracked by boat and plane. Linear home ranges did not differ in relation to sex but did vary among seasons, and the majority of movement occurred in spring. Seven of the 18 (39%) Lake Sturgeon left the river and were not detected in the river again during the study. Between 56% and 70% of subadult fish remaining in the river made definitive movements to, or near, known spawning locations when adult Lake Sturgeon were actively spawning. Our results suggest only a small proportion of subadult Lake Sturgeon in the Lake Winnebago population use the lower Wolf River, indicating that riverine sampling may not always be warranted when targeting subadults in large lake–river complexes. More information is needed on distribution of subadult Lake Sturgeon to develop sampling protocols for this population segment.