The influence of soybean row width and glyphosate application timing was determined on survival, biomass, and seed production of cohorts from a mixed population of Palmer amaranth and pusley species (Florida and Brazil pusley) along with soybean seed yield. The first Palmer amaranth and pusley cohort comprised plants that emerged from soybean planting through the V3 (3 wk after soybean emergence [WAE]) soybean stage (cohort 1). The second cohort comprised plants that emerged between the V3 to V6 (5 WAE) soybean stages (cohort 2), and the third cohort emerged after the V6 through the R2 soybean stage (cohort 3). Glyphosate at 840 g ae ha⁻¹ was applied at V3; V6; V3 and V6; and V3, V6, and R2 in rows either 19 or 97 cm wide. A nontreated control was included for comparison in each row width. Sequential glyphosate applications at V3 and V6 or at V3, V6, and R2 soybean stages resulted in 1 to 3% survival of cohort 1 compared with 23 to 28% survival after a single glyphosate application. Vegetative biomass production by cohort 1 accounted for 71% of the total pusley biomass produced in the nontreated plots. Cohort 1, 2, and 3 contributed 68, 31, and 1%, respectively, of the total 37,900 seeds m⁻² produced by pusley plants in nontreated plots. Delaying a glyphosate application to the V6 stage resulted in higher biomass and more than twice the seed produced from cohort 1 when compared with cohort 2. Glyphosate applied at V3 and V6 stages prevented pusley seed production from cohort 1, and an additional glyphosate application at the R2 stage prevented seed production from cohorts 2 and 3. No Palmer amaranth emergence occurred after the V6 soybean stage in either row width. A single glyphosate application at the V3 or V6 stage eliminated cohort 1 of Palmer amaranth in narrow rows. Palmer amaranth plants from cohort 1 in wide rows that survived the V3 glyphosate application produced 3.3 g m⁻² biomass and 600 seeds m⁻². Averaged over years and row widths, soybean yields after sequential glyphosate applications were 2,490 to 2,640 kg ha⁻¹ compared with 1,850 to 2,020 kg ha⁻¹ after a single glyphosate application at the V3 or V6 stage. This research confirms that sequential glyphosate applications are superior to a single application for minimizing pusley and Palmer amaranth survival, biomass, and seed production along with an improvement in soybean yields.