Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5.U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.