Previous studies of railroad employment in the nineteenth-century USA have focused on occupational mobility but remarkably little is known in quantitative terms about the geographical and inter-sectoral mobility of railroad workers in a regional or inter-regional context. Uniquely in the antebellum era among the four main trans-Appalachian railroads, which were the largest in the nation, the Baltimore and Ohio (B&O) published comprehensive payroll lists for several specific months between 1842 and 1857. Analysis of these payrolls in conjunction with city directories and manuscript schedules from the 1860 census has enabled reconstruction of key aspects of regional labor market dynamics linked to the railroad sector during this period. A number of important findings emerge, which both amplify and run counter to received wisdom. The most significant of these is that railroad workers fell largely into two groups regardless of skill level. The first group made the railroad a source of long-term employment, while the second used the railroad as a source of temporary employment either in between spells of agricultural labor locally or as a means of raising funds to move west and acquire land in newly settled districts closer to the frontier. This nineteenth-century tendency to revert to the agricultural sector runs counter to the expectations of twentieth-century human capital theory. The conventional post-bellum picture of large numbers of rootless ‘boomers’ gravitating westwards between railroad companies, is not in evidence on the B&O of the late 1850s. Over longer time-spans, there is some westward movement along the B&O itself, although this was partially counterbalanced by an eastward flow toward Baltimore from the interior. Shop-men had lower retention rates, especially at more westerly points, than the supposedly mobile trainmen, while railroad employees in generic trades, such as blacksmiths and machinists, were more likely to move than their non-railroad counterparts. This important finding suggests that generic workers should not be treated as homogeneous occupational groups, although such homogeneity is very largely implicit in the coding of available digital census datasets. Further to this, extensive construction work along the line of the B&O persisted long after operational service had commenced, and this provided additional opportunities for regular or intermittent railroad labor force participation. Overall, though it was not immune from competition for labor, the enormous size of the B&O compared to most manufacturing establishments of the time made it a key regulator of employment opportunity across the region that it served.